Silica adsorbent for removal of chlorophyll derivatives from triacylglycerol-based oils

ABSTRACT

The present invention relates to an adsorbent for treating an oil comprising a chlorophyll derivative. In particular, the present disclosure relates to an improved silica gel adsorbent for removing impurities, including chlorophyll derivatives and/or trace metals, from an oil, in particular triacylglycerol-based oils. The adsorbent comprises a silica gel treated with an alkali earth metal oxide, such as magnesium oxide, and has a pH of about 7 or greater and a water content of about 3 wt % or greater.

FIELD OF THE DISCLOSURE

The present disclosure relates to a process for treating an oilcomprising chlorophyll derivatives. In particular, the presentdisclosure relates to an improved process for removing impurities,including chlorophyll derivatives and/or trace metals, from an oil, andto improved silica based adsorbents for use in the process.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 26, 2019, isnamed ‘35893-513_SEQ_LIST.txt’ and is 36,864 bytes in size.

BACKGROUND OF THE DISCLOSURE

Crude triacylglycerol oils obtained from either pressing or solventextraction methods are a complex mixture of triacylglycerols,phospholipids, sterols, tocopherols, diacylglycerols, free fatty acids,trace metals, chlorophylls, beta-carotene, and other minor compounds. Itis desirable to remove the phospholipids, free fatty acids, tracemetals, chlorophylls, and beta-carotene in order to produce a qualityfully refined oil or a salad oil with a bland taste, light color, and along shelf life.

The removal of phospholipids generates the largest amount of neutral oillosses associated with the refining of triacylglycerol oils. The removalof chlorophylls generates the second largest amount of neutral oillosses associated with the refining of triacylglycerol-based oils.

Several different techniques may be used for phospholipid removal,including water degumming, enzyme assisted water degumming, aciddegumming, caustic refining, and enzymatic treatment.

Water degumming is usually applied to crude oils containing a highamount of hydratable phospholipids. Due to its mild characteristics, thephospholipids obtained can be used as lecithin (a natural emulsifier).The oil obtained from this technique is generally referred to in theindustry as being “degummed,” despite being only partially degummed.Since water degummed oil still contains high amounts of phospholipids,especially non-hydratable phospholipids, the use of other processtechniques, such as caustic refining or phospholipase A (PLA) enzymedegumming, can be required to produce a finished, high quality oilhaving high stability and low color.

In the water degumming process, water is added to crude oil with mixingto aid the hydration of the phospholipids present in the oil. Thehydration of the phospholipids or “gums” causes the gums to swell andagglomerate as a flocculent, which is subsequently separated from theremainder of the oil. The oil loss from water degumming processes may besignificant, with a negative impact in the overall economic balance onthe refined oil process cost.

Enzyme assisted water degumming is usually applied to crude oilscontaining a high amount of hydratable phospholipids, where the goal isto react all of the hydratable phospholipids and convert them intodiacylglycerols increasing the oil yield, while maintaining thenon-hydratable phospholipids in the oil. Enzymes utilized for thisprocess are Phospholipase C (PLC) and Phosphatidyl InositolPhospholipase (PI-PLC).

In the enzyme assisted water degumming process, water and PLCs are addedto crude oil with mixing. The enzymes are then allowed to react with thephospholipids in the oil with shear mixing to aid in the conversion ofphosphatidyl choline (PC), phosphatidyl ethanolamine (PE), and PI todiacylglycerols in the oil. The heavy phase (water, denature protein,and phosphor-compounds) has a specific gravity higher than that of theoil and may be separated by settling, filtration, or the industrialpractice of centrifugation. The enzyme assisted water degumming processremoves predominately only the hydratable phospholipids. The remainingphospholipids, measured as the salts of phosphatidic acid can be removedin subsequent processing operations.

Acid degumming is usually applied to crude oils when the goal is thetotal removal of phospholipids. The oil obtained is usually called“super-degummed” or “totally degummed” in the industry. Crude oil istreated with phosphoric acid or citric acid. The acid improves thehydrophilic nature of the non-hydratable phospholipids (NHPs), thusaiding in their removal. Water is then added to the acid-treated crudeoil, and the oil is mixed to aid the hydration of the phospholipids. Thehydration of the phospholipids or “gums” causes the gums to swell andagglomerate as a flocculent, which is subsequently removed. The aciddegumming process removes most of the phospholipids, but enough stillremain in the degummed oil to require additional processing. As in thewater degumming process, some of the oil is emulsified, and isconsidered a process loss, with the negative economic impact on theoverall economic balance of the refined oil process cost.

Caustic refining is usually applied to crude or water degummed oils whenthe goal is to remove all of the phospholipids and free fatty acids.Crude or water degummed oil is treated with phosphoric acid or citricacid. The acid improves the hydrophilic nature of the NHPs, thus aidingin their removal. A diluted sodium hydroxide solution is added to theacid-treated oil. The caustic solution neutralizes the free fatty acids(producing sodium soaps), neutralizes the excess acid, and with thesodium soaps created, assists in hydrating and emulsifying all theremaining phospholipids. The sodium hydroxide solution/oil is mixed andthen separated by settling, filtration, or industrially bycentrifugation. The caustic treated oil is then “washed” and centrifugedagain. The oil from the centrifuge is known as “Once Refined” and thewater is commonly known as “Wash Water”. For food applications, the“once refined” oil is usually submitted for bleaching and deodorizationto produce salad oil. An alternative to water washing is to treat thecaustic treated oil with an adsorbent silica gel and filter out theresidual soaps and phospholipids not removed in the initialcentrifugation.

“Enzymatic refining” or “enzymatic degumming” is used when the goal isthe total removal of phospholipids. Generally, enzymatic degummingtreatments of the prior art have been practiced on oils that have beendegummed previously by one of the other methods, typically waterdegumming. For food applications, the enzyme degummed oil issequentially submitted to bleaching and deodorization, a process knownin the industry as “physical refining.” Enzymatic degumming provides abetter oil yield than water, acid, or caustic degumming, with improvedeconomic results.

The enzymatic reaction changes the nature of the phospholipid, cleavingsome of the phospholipid parts. This reduces the phospholipids'emulsification properties, so that less oil is lost when the gums areseparated from the oil, thus saving oil. Enzymes exhibiting activitywith phospholipids are commonly called “phospholipases”. The types ofphospholipase are based on the position on the phospholipid molecule atwhich the enzyme reacts, and are known as PLA1, PLA2, PLC, and PLD.Different types of phospholipases will yield different compounds uponreacting with the phospholipids.

Commercial PLA1 enzymes with phospholipase activity are Lecitase® Ultraand QuaraLowP. Commercial PLA2 enzymes with phospholipase activity areRohalase Xtra and LysoMax. These products are known to yield polarlyso-phospholipids and polar fatty acids when mixed with degummed oilwith a 1-1.5% water citric acid-NaOH buffer at 4.5<pH<7.0 and 40°C.<T<55° C. The PLA1 selectively hydrolyzes the fatty acid opposite thephosphate functional group on the glycerol backbone and the PLA2selectively hydrolyzes the fatty acid in the center of the glycerolbackbone of the phospholipid. PLAs are non-selective for thephospholipids they react with.

The resulting reaction yields a lyso-phospholipid and a fatty acid. Thelyso-phospholipid molecule has lost one hydrophilic functional group,and the remaining alcohol group at the reaction site is hydrophilic. Nowwith two hydrophilic sites, the lyso-phospholipid molecule is watersoluble, and has lost its emulsification properties. The PLA1 or PLA2degumming process thus reduces refining losses by no longer removing anyneutral oil with the gums, and the only loss is the originalphospholipid molecule.

It is known in the art that PLC enzymes react with a phospholipid byselectively hydrolyzing the phosphate functional group. The resultingreaction yields a diacylglycerol (“DAG”) and a phosphatidic group. Thediacylglycerol molecule no longer has the phosphate functional group anddoes not need to be removed. The PLC degumming process reduces therefining loss by retaining the original phospholipid molecule, whileremoving only the phosphate functional group. However, PLC does notreact with all of the phospholipids present in the oil. Generally, PLCdoes not react with either phosphatidic acid (PA) or phosphatidylinositol (PI). A PI-PLC used in combination with PLC enables thereaction and removal of PC, PE, and Pl. Yet the non-hydratablephosphatides that remain in oil after water degumming. Thus, theenzymatic assisted water degumming treated oil must be further treatedwith caustic to remove the residual gums, or may further be treated withPLA1 or PLA2.

Triacylglycerol oils from oilseeds such as soybean and canola, and oilfruits, such as palm and algal source oils, contain chlorophyll.Chlorophyll is removed during many stages of the oil production process,including seed crushing, oil extraction, degumming, caustic treatmentand bleaching steps. In the last of these, the bleaching processresidual chlorophyll is removed to achieve acceptable levels. Thischlorophyll is typically removed from the oil in a bleaching processstep involving heating the oil and running it through an adsorbent toremove chlorophyll and other color-bearing compounds that impact theappearance and/or stability of the finished oil.

High level of chlorophyll pigments imparts undesirable color and induceoxidation of oil during storage leading to a deterioration of the oil.In the edible oil processing industry, a bleaching step is employed tolower chlorophyll levels to as low as 0.02 ppm to guarantee oil qualityin terms of color and organolepticity. This bleaching step increasesprocessing cost and reduces oil yield due to entrainment in thebleaching clay. The “spent” clay then must be disposed ofenvironmentally and is a hazardous material to transport due to thespontaneous combustion nature acid treated material and adsorbed oil,approximately 30% wt.

Chlorophyll is modified during oil processing into a derivative known aspheophytin, by the loss of the magnesium ion from the porphyrin(chlorine) ring (see FIG. 1 ). Typically, pheophytin is more abundant inoil during processing than chlorophyll. Pheophytin can be furtherdegraded into pyropheophytin (see Behavior of Chlorophyll Derivatives inCanola Oil Processing”, JAOCS, Vol. no. 9 September 1993, p. 837-841).Pyropheophytin is predominantly formed processing of vegetable oils (seee.g. ‘The lipid handbook’ ed. Frank D. Gunstone, John L. Harwood, AlbertJ. Dijkstra. 2007—3rd ed., p. 56). Chlorophyll, pheophytin andpyropheophytin occur in two forms the A and B form. The A component hasa methyl group at the C7 position. The B component has an aldehyde atthe C7 position.

The use of enzymes for the removal of pyropheophytin in vegetable oilsis known from WO2010/143149 and WO2013/160372. WO2010/143149 disclosesmethods for treating pyropheophytin-containing compositions usingenzymes capable of hydrolysing pyropheophytin derived for instance fromTriticum aestivum and Chlamydomonas reinhardtii. WO2013/160372 disclosesseveral chlorophyllase enzymes for instance from Arabidopsis thalianaand Triticum aestivum, which were able to convert pheophytin andpyropheophytin in oil.

Silicas have also been used as an adsorbent for removing impurities fromtriacylglycerol-based oils. Examples of silicas that have been used topurify oils include those described in U.S. Pat. Nos. 9,295,810;4,781,864; and 9,493,748. Such silicas, however, are not fully effectiveat removing impurities from oils, and undesirable levels of impurities,including colorants such as chlorophyll derivatives, may remain in theoils even after silica treatment.

There is thus a need for alternative silicas that are capable ofremoving impurities from triacylglycerol-based oils.

SUMMARY OF THE DISCLOSURE

In one aspect, the present disclosure is directed to a process fortreating an oil comprising a chlorophyll derivative, the processcomprising contacting the oil with an adsorbent comprising a silicatreated with an alkaline earth metal oxide, wherein the adsorbent has apH of about 7 or greater, including from about 7 to about 10, comprisesabout 0.1 wt. % or greater of alkaline earth metal oxide, such as MgO,on a dry basis, and has a water content of about 3 wt. % or greater, andpreferably, about 10 wt. % or greater, or from about 25 wt. % to about75 wt. %.

In one particular aspect, the present disclosure is directed to aprocess for treating an oil comprising a chlorophyll derivative, theprocess comprising contacting the oil with an adsorbent comprising asilica treated with an alkaline earth metal oxide, wherein the adsorbenthas a pH of from about 7 to about 10, and comprises from about 2.5 toabout 15 wt. %, or from about 5 to about 25 wt %, or from about 10 toabout 20 wt % of MgO, on a dry basis, and has a water content of fromabout 25 to about 75 wt. %.

In another aspect, the present disclosure is directed to a process fortreating an oil comprising a chlorophyll derivative, the processcomprising: contacting the oil with a polypeptide having decoloraseactivity, or a composition comprising the polypeptide, to produce adecolorase-treated oil, and contacting the decolorase-treated oil withan adsorbent comprising a silica treated with an alkaline earth metaloxide, wherein the adsorbent has a pH of about 7 or greater, e.g., fromabout 7 to about 10, comprises about 0.1 wt. % or greater of alkalineearth metal oxide, such as MgO, on a dry basis, and has a water contentof about 3 wt. % or greater, preferably, about 10 wt. % or greater, orfrom about 25 to about 75 wt. %.

In another aspect, the present disclosure is directed to a process fortreating an oil comprising a chlorophyll derivative, the processcomprising: contacting the oil with a polypeptide having decoloraseactivity, or a composition comprising the polypeptide, to produce adecolorase-treated oil, and contacting the decolorase-treated oil withan adsorbent comprising a silica treated with an alkaline earth metaloxide; wherein the adsorbent has a pH of from about 7 to about 10, andcomprises from about 2.5 to about 15 wt. %, or from about 5 to about 25wt %, or from about 10 to about 20 wt % of MgO, on a dry basis, and hasa water content of from about 25 to about 75 wt. %.

In another aspect, the present disclosure is directed to a process fortreating an oil comprising pyropheophytin, the process comprising:contacting the oil with a polypeptide having pyropheophytinase activity,or a composition comprising the polypeptide, wherein pyropheophytin isconverted into pyropheophorbide, and optionally wherein pheophytin isconverted into pheophorbide to produce a pyropheophytinase-treated oil,and contacting the pyropheophytinase-treated oil with an adsorbentcomprising a silica treated with an alkaline earth metal oxide, whereinthe adsorbent has a pH of about 7 or greater, e.g. from about 7 to about10, comprises about 0.1 wt. % or greater of alkaline earth metal oxide,such as MgO, on a dry basis, and has a water content of about 3 wt. % orgreater, preferably, about 10 wt. % or greater, or from about 25 toabout 75 wt. %.

In another aspect, the present disclosure is directed to a process fortreating an oil comprising pyropheophytin, the process comprising:contacting the oil with a polypeptide having pyropheophytinase activity,or a composition comprising the polypeptide, wherein pyropheophytin isconverted into pyropheophorbide, and optionally wherein pheophytin isconverted into pheophorbide to produce a pyropheophytinase-treated oil,and contacting the pyropheophytinase-treated oil with an adsorbentcomprising a silica treated with an alkaline earth metal oxide; whereinthe adsorbent has a pH of from about 7 to about 10, and comprises fromabout 2.5 to about 15 wt. %, or from about 5 to about 25 wt %, or fromabout 10 to about 20 wt % of MgO, on a dry basis, and has a watercontent of from about 25 to about 75 wt. %.

In another aspect, the present disclosure is directed to a process fortreating an oil comprising a chlorophyll derivative, the processcomprising: contacting the oil with a polypeptide having decoloraseactivity, or a composition comprising the polypeptide, to produce adecolorase-treated oil, and contacting the decolorase-treated oil withan adsorbent comprising a silica treated with an alkaline earth metaloxide, wherein the adsorbent has a pH of about 7 or greater, e.g. fromabout 7 to about 10, comprises about 0.1 wt. % or greater of alkalineearth metal oxide, such as MgO, on a dry basis, and has a water contentof about 3 wt. % or greater, preferably, about 10 wt. % or greater, suchas from about 25 to about 75 wt. %; and wherein the treatment reducesthe total concentration of chlorophyll derivatives in the composition byat least 5% by weight, compared to the total concentration ofchlorophyll derivatives in the composition prior to contact with theadsorbent.

In another aspect, the present disclosure is directed to a process fortreating an oil comprising a chlorophyll derivative, the processcomprising: contacting the oil with a polypeptide having decoloraseactivity, or a composition comprising the polypeptide, to produce adecolorase-treated oil, and contacting the decolorase-treated oil withan adsorbent comprising a silica treated with an alkaline earth metaloxide; wherein the adsorbent has a pH of from about 7 to about 10, andcomprises from about 2.5 to about 15 wt. %, or from about 5 to about 25wt %, or from about 10 to about 20 wt % of MgO, on a dry basis, and hasa water content of from about 25 to about 75 wt. %; and wherein thetreatment reduces the total concentration of chlorophyll derivatives inthe composition by at least 5% by weight, compared to the totalconcentration of chlorophyll derivatives in the composition prior tocontact with the adsorbent.

In another aspect, the polypeptide used in a process of the presentdisclosure is a polypeptide having pyropheophytinase activity, whereinthe polypeptide is selected from the group consisting of:

-   -   a. an isolated polypeptide which has at least 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at        least 99%, or 100% identity to amino acids 1 to 318 of SEQ ID        NO: 1; and,    -   b. a polypeptide encoded by a nucleic acid sequence that has at        least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98% or at least 99%, or 100% identity to the        nucleic acid sequence of SEQ ID NO: 2.

In one embodiment, the polypeptide used in a process of the presentdisclosure converts a chlorophyll substrate into a chlorophyll product.A chlorophyll substrate may be selected from the group consisting ofchlorophyll, pheophytin, pyropheophytin, and combinations thereof, andthe chlorophyll product may be selected from the group consisting ofchlorophyllide, pheophorbide, pyropheophorbide, and combinationsthereof.

In one aspect, the oil comprises pyropheophytin, the polypeptide haspyropheophytinase activity as disclosed herein, and the pyropheophytinis converted into pyropheophorbide.

In another aspect, the present disclosure is directed to an oil producedby a process disclosed herein.

In another aspect, the present disclosure is directed to improved silicabased adsorbents for use in the processes of the disclosure. Theadsorbents comprise an amorphous porous silica treated with an alkalineearth metal oxide, preferably magnesium oxide, in an amount sufficientto provide a pH of about 7 or greater and a water content of about 3 wt.% or greater in the final adsorbent.

Definitions

The term “control sequence” can be used interchangeably with the term“expression-regulating nucleic acid sequence”. The term as used hereinrefers to nucleic acid sequences necessary for and/or affecting theexpression of an operably linked coding sequence in a particular hostorganism, or in vitro. When two nucleic acid sequences are operablylinked, they usually will be in the same orientation and also in thesame reading frame. They usually will be essentially contiguous,although this may not be required. The expression-regulating nucleicacid sequences, such as inter alia appropriate transcription initiation,termination, promoter, leader, signal peptide, propeptide,prepropeptide, or enhancer sequences; Shine-Dalgarno sequence, repressoror activator sequences; efficient RNA processing signals such assplicing and polyadenylation signals; sequences that stabilizecytoplasmic mRNA; sequences that enhance translation efficiency (e.g.,ribosome binding sites); sequences that enhance protein stability; andwhen desired, sequences that enhance protein secretion, can be anynucleic acid sequence showing activity in the host organism of choiceand can be derived from genes encoding proteins, which are eitherendogenous or heterologous to a host cell. Each control sequence may benative or foreign (heterologous) to the nucleic acid sequence encodingthe polypeptide. When desired, the control sequence may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide. Control sequences maybe optimized to their specific purpose.

The term “chlorophyll derivatives” as used herein includes chlorophyllsubstrates and chlorophyll products. Chlorophyll substrates comprisechlorophyll, pheophytin and pyropheophytin. Chlorophyll productscomprise chlorophyllide, pheophorbide and pyropheophorbide. Chlorophyllderivatives comprise so-called a and b compounds.

The term “decolorase” (as well as variations thereof, including thephrase “a polypeptide having decolorase activity”), as used herein,means the polypeptide is capable of converting one or more chlorophyllsubstrate into a chlorophyll product. For instance, the polypeptide maybe capable of hydrolyzing chlorophyll into chlorophyllide; hydrolyzingpheophytin into pheophorbide; and/or hydrolyzing pyropheophytin intopyropheophorbide. The term “decolorase activity” thus may includechlorophyllase activity, pheophytinase activity, pyropheophytinaseactivity, or combinations thereof.

The term “hydrogel” is used herein to refer to a silica-based adsorbentthat has a water content of about 25 wt % or greater, and preferablyfrom about 25 to about 75 wt. %.

The term “amorphous” is used herein to mean a solid material whoseconstituent atoms, molecules, or ions are arranged in a random,non-ordered pattern that extends in all three directions, which may bedetermined by X-ray diffraction or differential scanning calorimetry.

The term “porous”, as used herein, refers to materials having aninternal porosity of 0.1 cc/g or greater as measured byBarrett-Joyner-Halenda (BJH) nitrogen porosimetry as described in DIN66134.

The term “treated”, as used herein with reference to treating with analkaline earth metal oxide, refers to the intimate mixing of silica andthe alkaline earth metal oxide under high shear conditions.

The term “particle surface area” is defined as meaning a particlesurface area as measured by the Brunauer Emmet Teller (BET) nitrogenadsorption method.

The phrase “median particle size” refers to median particle size (D50,which is a volume distribution with 50 volume percent of the particlesbeing smaller than this number and 50 volume percent of the particlesbeing bigger than this number in size), measured by dynamic lightscattering when the particles are slurried in water or an organicsolvent such as acetone or ethanol.

The term “triacylglycerol-based oil” refers to an oil comprisingtriacylglycerol.

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription, posttranscriptional modification, translation, post-translationalmodification, and secretion.

An expression vector comprises a polynucleotide coding for apolypeptide, operably linked to the appropriate control sequences (suchas a promoter, RBS/Shine Delgado and transcriptional and translationalstop signals) for transcription and/or translation in vitro, or in thehost cell, of the polynucleotide.

The expression vector may be any vector (e.g., a plasmid or virus),which can be conveniently subjected to recombinant DNA procedures andcan bring about the expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thecell into which the vector is to be introduced. The vectors may belinear or closed circular plasmids. The vector may be an autonomouslyreplicating vector, i.e. a vector, which exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a plasmid, an extra-chromosomal element, amini-chromosome, or an artificial chromosome. Alternatively, the vectormay be one which, when introduced into the host cell, is integrated intothe genome and replicated together with the chromosome(s) into which ithas been integrated. The integrative cloning vector may integrate atrandom or at a predetermined target locus in the chromosomes of the hostcell. The vector system may be a single vector or plasmid or two or morevectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon.

A host cell as defined herein is an organism suitable for geneticmanipulation and one which may be cultured at cell densities useful forindustrial production of a target product, such as a polypeptideaccording to the present invention. A host cell may be a host cell foundin nature or a host cell derived from a parent host cell after geneticmanipulation or classical mutagenesis. Advantageously, a host cell is arecombinant host cell.

A host cell may be a prokaryotic, archaebacterial, or eukaryotic hostcell. A prokaryotic host cell may be, but is not limited to, a bacterialhost cell. A eukaryotic host cell may be, but is not limited to, ayeast, a fungus, an amoeba, an alga, a plant, an animal, or an insecthost cell.

The term “heterologous” as used herein refers to nucleic acid or aminoacid sequences not naturally occurring in a host cell. In other words,the nucleic acid or amino acid sequence is not identical to thatnaturally found in the host cell.

A nucleic acid or polynucleotide sequence is defined herein as anucleotide polymer comprising at least 5 nucleotide or nucleic acidunits. A nucleotide or nucleic acid refers to RNA and DNA. The terms“nucleic acid” and “polynucleotide sequence” are used interchangeablyherein.

A “peptide” refers to a short chain of amino acid residues linked by apeptide (amide) bonds. The shortest peptide, a dipeptide, consists of 2amino acids joined by single peptide bond.

The term “polypeptide” refers to a molecule comprising amino acidresidues linked by peptide bonds and containing more than five aminoacid residues. The term “protein” as used herein is synonymous with theterm “polypeptide” and may also refer to two or more polypeptides. Thus,the terms “protein” and “polypeptide” can be used interchangeably.Polypeptides may optionally be modified (e.g., glycosylated,phosphorylated, acylated, farnesylated, prenylated, sulfonated, and thelike) to add functionality. Polypeptides exhibiting activity in thepresence of a specific substrate under certain conditions may bereferred to as enzymes. It will be understood that, as a result of thedegeneracy of the genetic code, a multitude of nucleotide sequencesencoding a given polypeptide may be produced.

An “isolated nucleic acid fragment” is a nucleic acid fragment that isnot naturally occurring as a fragment and would not be found in thenatural state.

The term “isolated polypeptide” as used herein means a polypeptide thatis removed from at least one component, e.g. other polypeptide material,with which it is naturally associated. The isolated polypeptide may befree of any other impurities. The isolated polypeptide may be at least50% pure, e.g., at least 60% pure, at least 70% pure, at least 75% pure,at least 80% pure, at least 85% pure, at least 80% pure, at least 90%pure, or at least 95% pure, 96%, 97%, 98%, 99%, 99.5%, 99.9% asdetermined by SDS-PAGE or any other analytical method suitable for thispurpose and known to the person skilled in the art. An isolatedpolypeptide may be produced by a recombinant host cell.

The term “promoter” is defined herein as a DNA sequence that binds RNApolymerase and directs the polymerase to the correct downstreamtranscriptional start site of a nucleic acid sequence to initiatetranscription. A promotor sequence may be native of or heterologousrelative to the nucleic acid sequence encoding the polypeptide.

The term “recombinant” when used in reference to a cell, nucleic acid,protein or vector, indicates that the cell, nucleic acid, protein orvector, has been modified by the introduction of a heterologous nucleicacid or protein or the alteration of a native nucleic acid or protein,or that the cell is derived from a cell so modified. Thus, for example,recombinant cells express genes that are not found within the native(non-recombinant) form of the cell or express native genes that areotherwise abnormally expressed, under expressed or not expressed at all.The term “recombinant” is synonymous with “genetically modified” and“transgenic”.

The terms “sequence identity” and “sequence homology” are usedinterchangeable herein. For the purpose of this invention, it is definedhere that in order to determine the percentage of sequence homology orsequence identity of two amino acid sequences or of two nucleic acidsequences, the sequences are aligned for optimal comparison purposes. Inorder to optimize the alignment between the two sequences gaps may beintroduced in any of the two sequences that are compared. Such alignmentcan be carried out over the full length of the sequences being compared.Alternatively, the alignment may be carried out over a shorter length,for example over about 20, about 50, about 100 or more nucleicacids/bases or amino acids. The sequence identity is the percentage ofidentical matches between the two sequences over the reported alignedregion. The percent sequence identity between two amino acid sequencesor between two nucleotide sequences may be determined using theNeedleman and Wunsch algorithm for the alignment of two sequences.(Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453).Both amino acid sequences and nucleotide sequences can be aligned by thealgorithm. The Needleman-Wunsch algorithm has been implemented in thecomputer program NEEDLE. For the purpose of this invention the NEEDLEprogram from the EMBOSS package was used (version 2.8.0 or higher,EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice,P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp 276-277,http://ennboss.bioinformatics.nl/). For protein sequences EBLOSUM62 isused for the substitution matrix. For nucleotide sequence, EDNAFULL isused. The optional parameters used are a gap-open penalty of 10 and agap extension penalty of 0.5. The skilled person will appreciate thatall these different parameters will yield slightly different results butthat the overall percentage identity of two sequences is notsignificantly altered when using different algorithms.

After alignment by the program NEEDLE as described above the percentageof sequence identity between a query sequence and a sequence of theinvention is calculated as follows: Number of corresponding positions inthe alignment showing an identical amino acid or identical nucleotide inboth sequences divided by the total length of the alignment aftersubtraction of the total number of gaps in the alignment. The identityas defined herein can be obtained from NEEDLE by using the NOBRIEFoption and is labeled in the output of the program as“longest-identity”.

The nucleic acid and protein sequences of the present disclosure canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, word length=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See the homepage of the NationalCenter for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

A “synthetic molecule”, such as a synthetic nucleic acid or a syntheticpolypeptide is produced by in vitro chemical or enzymatic synthesis. Itincludes, but is not limited to, variant nucleic acids made with optimalcodon usage for host organisms of choice.

A synthetic nucleic acid may be optimized for codon use, preferablyaccording to the methods described in WO2006/077258 and/or WO2008000632,which are herein incorporated by reference. WO2008/000632 addressescodon-pair optimization. Codon-pair optimization is a method wherein thenucleotide sequences encoding a polypeptide that have been modified withrespect to their codon-usage, in particular the codon-pairs that areused, are optimized to obtain improved expression of the nucleotidesequence encoding the polypeptide and/or improved production of theencoded polypeptide. Codon pairs are defined as a set of two subsequenttriplets (codons) in a coding sequence. Those skilled in the art willknow that the codon usage needs to be adapted depending on the hostspecies, possibly resulting in variants with significant homologydeviation from SEQ ID NO: 1, but still encoding the polypeptideaccording to the invention.

As used herein, the terms “variant”, “derivative”, “mutant” or“homologue” can be used interchangeably. They can refer to eitherpolypeptides or nucleic acids. Variants include substitutions,insertions, deletions, truncations, transversions, and/or inversions, atone or more locations relative to a reference sequence. Variants can bemade for example by site-saturation mutagenesis, scanning mutagenesis,insertional mutagenesis, random mutagenesis, site-directed mutagenesis,and directed-evolution, as well as various other recombinationapproaches known to a skilled person in the art. Variant genes ofnucleic acids may be synthesized artificially by known techniques in theart.

Detailed Description of the Disclosure

The present disclosure relates to processes for removing impurities,including phosphorus-containing compounds such as phosphorus gums, soap,trace metals, chlorophyll derivatives, free fatty acids, and the like,from oils, and in particular from triacylglycerol-based oils. Moreparticularly, the present disclosure is directed to adsorbentscomprising an amorphous, porous silica that has been treated with analkaline earth metal oxide, and the use of such adsorbents, to removeimpurities from oils.

It has now been discovered that treating a porous, amorphous silicahaving a specified water content with alkaline earth metals such asmagnesium, and in particular with alkaline earth metal oxides, such asmagnesium oxide, results in an adsorbent having an improved ability toremove impurities, such as chlorophyll derivatives and trace metals,from oils, as compared to previously known silicas. It has further beendiscovered that alkaline earth metal oxide-treated silica adsorbentshaving a pH of about 7 or greater, and preferably from about 7 to about10, are capable of removing more impurities (e.g., trace metals,chlorophyll derivatives, etc.) from triacylglycerol-based oils than areprior adsorbents based on other types of silicas, such as xerogel, oracidic hydrogels.

Thus, in one aspect, the present disclosure is directed to a process fortreating an oil comprising a chlorophyll derivative, the processcomprising contacting the oil with an adsorbent, wherein the adsorbentcomprises a silica, and in particular a porous, amorphous silica, thathas been treated with an alkaline earth metal oxide, such as MgO,wherein the adsorbent has a pH of about 7 or greater, and a watercontent of about 3 wt. % or greater, or preferably about 10 wt. % orgreater, or from about 25 to about 75 wt. %. In one particularembodiment, the adsorbent is a hydrogel, and has a pH of from about 7 toabout 10, and a water content of from about 25 to about 75 wt. %, andcomprises from about 2.5 to about 15 wt. %, or from about 5 to about 25wt %, or from about 10 to about 20 wt % of MgO, on a dry basis.

In one embodiment, the adsorbent comprises a porous, amorphous silicatreated with an alkaline earth metal oxide in an amount sufficient toprovide about 0.1 wt. % or greater, and more typically, from about 1 wt.% to about 40 wt. %, or from about 5 wt. % to about 25 wt. %, or fromabout 10 to about 20 wt %, or from about 2.5 wt. % to about 15 wt. %, ofalkali earth metal oxide, on a dry basis. In one particular embodiment,the silica is treated with magnesium oxide (MgO), and the amorphoussilica is a gel. In such embodiments, the adsorbent comprises about 0.1wt % or greater of MgO, or from about 1 wt. % to about 40 wt. %, or fromabout 2.5 wt. % to about 15 wt. %, or from about 5 wt. % to about 25 wt.%, or from about 5 wt. % to about 15 wt. %, or from about 10 wt. % toabout 20 wt. %, or from about 10% to about 15 wt. % of MgO, on a drybasis. In one particular embodiment, the adsorbent comprises from about10 wt. % to about 20 wt. % of MgO.

In some embodiments, the adsorbent of the present disclosure has a molarratio of MgO to SiO₂ of from about 1:3.8 to about 1:26, including fromabout 1:12.77 to about 1:3.3, or from about 1:12.77 to about 1:4.89, orfrom about 1:8.09 to about 1:4.90, or from about 1:5.44 to about 1:4.89.In one particular embodiment, the adsorbent has a molar ratio of fromabout 1:5.44 to about 1:4.89 of MgO to SiO₂.

The adsorbent of the present disclosure may have a water content of atleast about 3 wt. %, and more typically, about 10 wt. % or greater,about 20 wt. % or greater, or even about 55 wt. % or greater. In certainembodiments, the adsorbents of the present disclosure are advantageouslyprepared from silica hydrogels, and have a water content of from about25 wt. % to about 75 wt. %. In other embodiments, the adsorbents have awater content of from about 30 wt. % to about 65 wt. %, or from about 40wt. % to about 70 wt. %, or from about 50 wt. % to about 65 wt. %, orfrom about 55 wt. % to about 67 wt. %, or from about 58 wt. % to about65 wt. %.

The adsorbents of the present disclosure have a pH of about 7 orgreater, including from about 7 to about 10, or from about 7.5 to about9.7, or from about 8.0 to about 9.5, or from about 8.0 to about 9.0, orfrom about 8.2 to about 9.3. As discussed herein, and as demonstrated inthe examples, the adsorbents of the present disclosure have beendiscovered to be superior at removing impurities from oils, as comparedto prior adsorbents based on acidic hydrogels.

The adsorbent of the present disclosure may have a median particle sizeof from about 0.1 to about 2000 microns, including from about 1 to about1000 microns, or from about 2 to about 500 microns, or from about 5 toabout 50 microns. In one embodiment, the adsorbent has a median particlesize of from about 10 to about 30 microns.

The adsorbents of the present disclosure may have a surface area ofabout 50 m²/g or greater, including about 300 m²/g or greater, or about650 m²/g or greater. In some embodiments, the adsorbent of the presentdisclosure has a surface area of from about 50 m²/g to about 800 m²/g,or from about 300 m²/g to about 700 m²/g, as determined by BET surfacemeasurement.

The adsorbent of the present disclosure may have a pore volume of about0.1 cc/g or great, preferably about 0.4 cc/g or greater. In someembodiments, the pore volume of the adsorbent may range from about 0.2cc/g to about 2.0 cc/g, or from about 0.7 cc/g to about 2.0 cc/g, asdetermined by nitrogen porosimetry.

In one particular embodiment, the adsorbent of the present disclosurecomprises an amorphous silica gel that has been treated with magnesiumoxide (MgO), and that has a pH of from about 7 to about 10, andcomprises from about 2.5 wt. % to about 15 wt. % MgO, on a dry basis,and has a water content of from about 25 wt. % to about 75 wt. %. Moreparticularly, in one embodiment, the adsorbent of the present disclosurecomprises an amorphous silica gel that has been treated with MgO, andthat has a pH of from about 8 to about 9, comprises from about 10 wt. %to about 20 wt. % of MgO, on a dry basis, and has a water content offrom about 50 wt. % to about 65 wt. %.

Suitable silicas that can be used to prepare the adsorbents of thepresent disclosure include amorphous porous silica gels and precipitateshaving a moisture content of about 3 wt. % or greater in the pores ofthe silica. In one embodiment, the amorphous silica is a “hydrogel”having a moisture content of about 25 wt. % or greater, preferablygreater than 40 wt. %, most preferably greater than 50 wt. % and evenmore preferably, from about 30 to about 65 wt. % moisture. In someembodiments, suitable silica hydrogels include, but are not limited to,commercially available silica gels, such as TRISYL® silica and TRISYL®300 (W.R. Grace & Co.-Conn., Columbia, Md.).

In another embodiment, a substantially moisture-free silica may be usedas a starting silica. By “substantially water free” is meant that thesilica used to prepare the adsorbent has less than about 15% moisture.The substantially water-free silica is subsequently hydrated bycontacting the gel with a sufficient amount of water to provide thedesired moisture content, i.e., about 30 wt. % or greater, preferably inthe pores of the silica prior to combining with the alkaline earth metaloxide. The amount of water to be added correlates to the pore volume ofthe specific silica used, and can readily be determined by those skilledin the art. Non-limiting descriptions of the preparation of silicassuitable for use in the processes of the present disclosure are setforth in the examples.

Suitable alkaline earth metal oxide useful to prepare the adsorbents ofthe disclosure include, but are not limited to, magnesium oxide, calciumoxide, strontium oxide, barium oxide, beryllium oxide or combinationsthereof. Preferably, the alkaline earth metal oxide is magnesium oxide.

The adsorbents of the present disclosure may be prepared by physicallyblending, preferably under high shear conditions, the desired amount ofan alkaline metal oxide powder with the amorphous silica gel having thedesired water content. The blending is conducted for a time and atemperature sufficient to provide free-flowing powder. Preferably, theblending is conducted at a temperature ranging from about roomtemperature to about 100° C. and for a time sufficient to obtain ahomogeneous mixture, e.g. about 1 sec or greater. The final adsorbent ofthe invention comprises an alkaline earth metal oxide treated silica gelhaving a moisture content of about 3 wt. % or greater and a pH of about7 or greater. In one embodiment the final adsorbent is a free-flowingpowder provided by W.R. Grace & Co.-Conn under the product designationSP-2115 which product contains from about 10 to about 13 wt. % magnesiumoxide, on a dry basis, a water content of from about 50 to about 65% anda pH of about 8.2 to about 9.3.

In general, the processes of the present disclosure comprise contactingthe oil with an adsorbent of the present disclosure in an amounteffective to remove impurities from the oil. In a non-limitingembodiment, the oil is contacted with the adsorbent of the presentdisclosure in an amount of about 10 wt. % or less, based on the weightof the oil. In other embodiments, the oil is contacted with theadsorbent in an amount of from about 0.01 wt. % to about 10 wt. %, or inan amount of from about 0.1 wt. % to about 8 wt. %, or in an amount ofabout 0.1 wt. % to about 5 wt. %, or in an amount of about 0.1 wt. % toabout 1 wt. %, or in an amount of about 0.1 wt. % to about 0.5 wt. %, orin an amount of about 0.1 wt. % to about 0.4 wt. %, or in an amount ofabout 0.1 wt. % to about 0.3 wt. %, or in an amount of about 0.1 wt. %to about 0.2 wt. %, based on the weight of the oil.

In certain embodiments, the oil may be contacted with an adsorbent ofthe present disclosure at a temperature of less than about 100° C., ormore typically, at a temperature of from about 60° C. to less than about100° C., including at a temperature of about 80° C. In some embodiments,the oil may be contacted with an adsorbent of the present disclosureunder vacuum at a temperature of less than about 110° C., such as at atemperature of from about 70° C. to about 130° C., or at a temperatureof about 100° C. In one embodiment, the vacuum may be from about 50 toabout 700 mbar, and more typically is about 100 mbar. In one embodiment,the oil is contacted with the adsorbent of the present disclosure undervacuum of about 100 mbar at a temperature of about 100° C. In anotherembodiment, the oil is contacted with the adsorbent of the presentdisclosure at a temperature of from about 60° C. to less than about 100°C., or at a temperature of about 80° C., followed by application of avacuum of from about 50 to about 700 mbar, and more typically about 100mbar, and an increase in temperature to about 70° C. to about 130° C. Inone particular embodiment, the oil is contacted with an adsorbent of thepresent disclosure at a temperature of about 80° C., followed byapplication of a vacuum of about 100 mbar, and an increase intemperature to about 100° C. In some embodiments, the adsorbent iscontacted with the oil for from about 5 to about 240 minutes, includingfrom about 15 to about 60 minutes. Following treatment, the silica maybe removed from the oil using any suitable technique, includingfiltration.

Impurities that may be removed from the oils using the processes of thepresent disclosure include, but are not limited to,phosphorus-containing compounds, including phosphorus gums, soap, tracemetals (such as, but not limited to, sodium, potassium, magnesium,calcium, iron, aluminum, and lead), chlorophyll substrates (includingchlorophyll, pheophytin, pyropheophytin), chlorophyll derivatives(including chlorophyllide, pheophorbide, and pyropheophorbide), and freefatty acids (FFA).

The treatment of the oil with an adsorbent of the present disclosure ashereinabove described provides an oil which meets acceptable standardsfor the trade and transportation of edible oils, including cooking oils.Such standards include those of the National Institute of OilseedProducts (NIOP), the American Oil Chemists Society, and the ISO.

Oils that may be treated using the processes of the present disclosureinclude, but are not limited to, a triacylglycerol-based oil selectedfrom the group consisting of canola oil, castor oil, coconut oil,coriander oil, corn oil, cottonseed oil, hazelnut oil, hempseed oil,linseed oil, mango kernel oil, meadowfoam oil, neat's foot oil, oliveoil, palm oil, palm kernel oil, palm olein, peanut oil, rapeseed oil,rice bran oil, safflower oil, sasanqua oil, sesame oil, soybean oil,sunflower seed oil, tall oil, tsubaki oil, vegetable oil, and an oilfrom algae. In one embodiment, the oil is an oil from algae.

The adsorbent of the present disclosure may be used to remove impuritiesfrom oils at a variety of stages during oil processing. For instance,the oil to be treated may be selected from the group consisting of acrude non-degummed oil, a degummed oil, a caustic refined oil, a causticrefined and water washed oil, or a water degummed oil. In oneembodiment, the oil is a crude oil, and the process comprises contactinga crude oil with a silica of the present disclosure.

In another embodiment, the oil is first subjected to decolorasetreatment prior to contacting with the adsorbent. For instance, in oneembodiment, the process comprises contacting an oil comprising achlorophyll derivative with a polypeptide having decolorase activity, orwith a composition comprising the polypeptide, to produce adecolorase-treated oil, and contacting the decolorase-treated oil withan adsorbent of the present disclosure. Examples of suitablepolypeptides having decolorase activity that may be used in theprocesses of the present disclosure include, but are not limited to,those polypeptides discussed in detail hereinafter. Suitable processesfor producing decolorase-treated oil, as well as various oil processingmethods, are also described in detail hereinafter.

Treatment of the oil with an adsorbent of the present disclosureadvantageously reduces the level of impurities relative to the level ofimpurities in the oil prior to contact with the adsorbent. For example,contacting an oil (including a decolorase-treated oil) with a silica ofthe present disclosure may reduce the total concentration of chlorophyllderivatives (including chlorophyll substrates and/or chlorophyllproducts) in the oil by at least 5%, at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 99%, or by 100% byweight, compared to the total concentration of chlorophyll derivatives(by weight) present in the oil prior to contact with the silica.

In another embodiment, the chlorophyll derivative in the oil comprisespyropheophytin, and contacting the oil (including a decolorase-treatedoil) with an adsorbent of the present disclosure may reduce the totalconcentration of pyropheophytin in the oil by at least 5%, at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, or by100% by weight, compared to the total concentration of pyropheophytin(by weight) present in the oil prior contact with the adsorbent.

In another embodiment, the chlorophyll derivative in the oil comprisespheophytin, and contacting the oil (including a decolorase-treated oil)with an adsorbent of the present disclosure may reduce the totalconcentration of pheophytin in the oil by at least 5%, at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, or by100% by weight, compared to the total concentration of pheophytin (byweight) present in the oil prior to contact with the silica.

In another embodiment, the chlorophyll derivative in the oil compriseschlorophyll, and contacting the oil (including a decolorase-treated oil)with a silica of the present disclosure reduces the total concentrationof chlorophyll in the oil by at least 5%, at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 99%, or by 100% byweight, compared to the total concentration of chlorophyll (by weight)present in the oil prior to prior to contact with the silica.

In another embodiment, contacting an oil (including a decolorase-treatedoil) with a silica-based adsorbent of the present disclosure may reducethe total concentration of trace metal impurities (such as, but notlimited to, sodium, potassium, magnesium, calcium, iron, aluminum, andlead), in the oil by at least 5%, at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 99%, or by 100% by weight,compared to the total concentration of trace metal impurities (byweight) present in the oil prior to contact with the adsorbent.

In another embodiment, an oil comprising a chlorophyll derivative, andin particular a chlorophyll substrate, is contacted with a polypeptidehaving decolorase activity, or with a composition comprising thepolypeptide, to produce a decolorase-treated oil. In one suchembodiment, the chlorophyll substrate comprises pyropheophytin, thedecolorase treatment converts at least a portion of the pyropheophytininto pyropheophorbide, and contacting the decolorase-treated oil with anadsorbent of the present disclosure reduces the total concentration ofpyropheophorbide in the decolorase-treated oil by at least 5%, at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least99%, or by 100% by weight, compared to the total concentration ofpyropheophorbide (by weight) present in the decolorase-treated oil priorcontact with the adsorbent. In another embodiment, the chlorophyllsubstrate comprises pheophytin, the decolorase treatment converts atleast a portion of the pheophytin into pheophorbide, and contacting thedecolorase-treated oil with an adsorbent of the present disclosurereduces the total concentration of pheophorbide in the oil by at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 99%, or by 100% by weight, compared to the totalconcentration of pheophorbide (by weight) present in thedecolorase-treated oil prior to contact with the adsorbent. In anotherembodiment, the chlorophyll substrate comprises chlorophyll, thedecolorase treatment converts at least a portion of the chlorophyll intochlorophyllide, and contacting the decolorase-treated oil with anadsorbent of the present disclosure reduces the total concentration ofchlorophyllide in the decolorase-treated oil by at least 5%, at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least99%, or by 100% by weight, compared to the total concentration ofchlorophyllide (by weight) present in the decolorase-treated oil priorto prior to contact with the adsorbent.

In one embodiment, the process of the present disclosure furthercomprises contacting the oil or the decolorase-treated oil with anadditional enzyme selected from the group consisting of a phospholipase,a pheophytinase, a pyropheophytinase, a pheophorbidase, achlorophyllase, and combinations thereof.

In another embodiment, the present disclosure is directed to a processfor treating an oil comprising pyropheophytin, the process comprisingcontacting the oil with a polypeptide having pyropheophytinase activity,or with a composition comprising the polypeptide, wherein pyropheophytinis converted into pyropheophorbide, and optionally where pheophytin isconverted into pheophorbide, to produce a pyropheophytinase-treated oil,and contacting the pyropheophytinase treated oil with an adsorbent ofthe present disclosure.

Polypeptides

Any suitable polypeptide having decolorase activity may be used in theprocesses of the present disclosure. In one particular embodiment, thepolypeptide is a polypeptide having pyropheophytinase activity, and isselected from the group consisting of:

-   -   a. an isolated polypeptide which has at least 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at        least 99%, or which has 100% identity to amino acids 1 to 318 of        SEQ ID NO: 1; and,    -   b. a polypeptide encoded by a nucleic acid sequence that has at        least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, or 99%, or which has 100% identity to the        nucleic acid sequence of SEQ ID NO: 2.

A polypeptide having pyropheophytinase activity may be a polypeptidewhich has at least 80% identity to amino acids 1 to 318 of SEQ ID NO: 1.A polypeptide as disclosed herein may have at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%identity to amino acids 1 to 318 of SEQ ID NO: 1. A polypeptide asdisclosed herein may have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to aminoacids 1 to 318 of SEQ ID NO: 1. A polypeptide having pyropheophytinaseactivity as disclosed herein may comprise, or contain, or consist ofamino acids 1 to 318 of SEQ ID NO: 1. A polypeptide havingpyropheophytinase activity may comprise, or contain, or consist of aminoacids 2 to 318 of SEQ ID NO: 1. Surprisingly, it was found that apolypeptide which has at least 80% identity to amino acids 1 to 318, orto amino acids 2 to 318 of SEQ ID NO: 1 comprises pyropheophytinaseactivity.

A polypeptide having pyropheophytinase activity hydrolysespyropheophytin into pyropheophorbide (see also FIG. 1 ). A polypeptidehaving pyropheophytinase activity as disclosed herein preferablyhydrolyses pyropheophytin a and pyropheophytin b into theirpyropheophorbide a and b compounds. Accordingly, pyropheophytinaseactivity can be determined by the formation of pyropheophorbide.

A polypeptide as disclosed herein may further comprise pheophytinaseactivity. A polypeptide having pheophytinase activity hydrolysespheophytin into pheophorbide. Preferably a polypeptide as disclosedherein hydrolyses pheophytin a and/or pheophytin b into their respectivepheophorbide compounds. Accordingly, pheophytinase activity can bedetermined by the formation of pheophorbide.

A polypeptide as disclosed herein having pyropheophytinase activity mayalso comprise chlorophyllase activity. A polypeptide havingchlorophyllase activity hydrolyses the conversion of chlorophyll intochlorophyllide. Preferably a polypeptide as disclosed herein hydrolyseschlorophyll a and/or chlorophyll b into their respective chlorophyllidecompounds.

In one embodiment, a polypeptide as disclosed herein haspyropheophytinase activity, pheophytinase activity, and chlorophyllaseactivity.

Determination of pyropheophytin, pheophytin, chlorophyll and thereaction products pyropheophorbide, pheophorbide, chlorophyllide can beperformed by HPLC as disclosed in the Examples.

A polypeptide may be derivable from any suitable origin, for instancefrom plant, algae or cyanobacteria. A polypeptide as disclosed hereinmay be derived from plant, for instance from Hordeum sp., or Triticumsp., for instance Hordeum vulgare or Triticum aestivum. A polypeptide asdisclosed herein may also be generated using standard moleculartechniques e.g. de novo synthesis.

A polypeptide having decolorase activity such as pyropheophytinaseactivity as disclosed herein may be an isolated, a pure, recombinant,synthetic or a variant polypeptide. A polypeptide as disclosed hereinmay be purified. Purification of proteins can be performed by severalmethods known to a person skilled in the art.

A variant polypeptide of a polypeptide having pyropheophytinase activityas disclosed herein may be a polypeptide that has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least99% identity to amino acids 1 to 318 of SEQ ID NO: 1, or to amino acids2 to 318 of SEQ ID NO:1.

A polypeptide having pyropheophytinase activity as disclosed herein maybe a polypeptide, for instance a variant polypeptide, which, whenaligned with an amino acid sequence according to SEQ ID NO: 1 comprisesa substitution, deletion and/or insertion at one or more amino acidpositions as compared to SEQ ID NO: 1. For instance, a polypeptide asdisclosed herein may be a polypeptide, which when aligned with apolypeptide of SEQ ID NO:1 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11or 12, or more amino substitutions, deletions and/or insertions ascompared to SEQ ID NO: 1, whereby the polypeptide still has the activityor function of a polypeptide as disclosed herein. The skilled personwill appreciate that these minor amino acid changes in a polypeptide asdisclosed herein may be present (for example naturally occurringmutations) or made (for example using r-DNA technology) without loss ofthe protein function or activity. In case these mutations are present ina binding domain, active site, or other functional domain of thepolypeptide a property of the polypeptide may change but the polypeptidemay keep its activity. In case a mutation is present which is not closeto the active site, binding domain, or other functional domain, lesseffect may be expected.

A polypeptide as disclosed herein may be encoded by any suitablepolynucleotide sequence, as long as the polypeptide exhibitspyropheophytinase activity as disclosed herein. Typically, apolynucleotide sequence encoding a polypeptide having pyropheophytinaseactivity as disclosed herein is a codon optimized sequence, or a codonpair optimized sequence for expression of the polypeptide in aparticular host cell.

Compositions

The processes of the present disclosure may further comprise contactingan oil with a composition comprising a polypeptide having decoloraseactivity, such as disclosed herein.

Such a composition may comprise a carrier, an excipient, or othercompounds. Typically, a composition, or a formulation, comprises acompound with which a polypeptide having decolorase activity (includingpyropheophytinase activity) may be formulated. Suitable formulationsinclude liquid formulations, such as emulsions, suspensions andsolutions, pastes, gels, granules and freeze-dried or spray-driedpowders.

An excipient as used herein is an inactive substance formulatedalongside with a polypeptide as disclosed herein, for instance sucroseor lactose, glycerol, sorbitol or sodium chloride. A compositioncomprising a polypeptide as disclosed herein may be a liquid compositionor a solid composition. A liquid composition usually comprises water.When formulated as a liquid composition, the composition usuallycomprises components that lower the water activity, such as glycerol,sorbitol or sodium chloride (NaCl). A solid composition comprising apolypeptide as disclosed herein may comprise a granulate comprising thepolypeptide or the composition comprises an encapsulated polypeptide inliquid matrices like liposomes or gels like alginate or carrageenans.There are many techniques known in the art to encapsulate or granulate apolypeptide or enzyme (see for instance G. M. H. Meesters,“Encapsulation of Enzymes and Peptides”, Chapter 9, in N. J. Zuidam andV. A. Nedovio (eds.) “Encapsulation Technologies for Active FoodIngredients and food processing” 2010).

A composition as disclosed herein may also comprise a carrier comprisinga polypeptide as disclosed herein. For instance, a polypeptide asdisclosed herein can be immobilized on silica. A polypeptide asdisclosed herein may be bound or immobilized to a carrier by knowntechnologies in the art.

A composition comprising a polypeptide having decolorase activity(including pyropheophytinase activity) as disclosed herein may compriseone or more further enzymes, for instance a lipase, such asphospholipase, for instance phospholipase A, B and/or C, achlorophyllase, pheophytinase and/or a pyropheophytinase. A furtherenzyme may be a phospholipase C (PLC), a phosphatidyl-inositol PLCand/or a phospholipase A, such as a phospholipase A1 or a phospholipaseA2.

A composition comprising a polypeptide having decolorase activity(including pyropheophytinase activity) as disclosed herein may comprisecell fractions for instance cell fractions from a host cell wherein thepolypeptide having decolorase activity has been produced. Cell fractionsmay be generated by various methods for instance after disruption of thehost cell by sonification and/or use of glass beads.

The present disclosure also relates to a process for preparing acomposition comprising a polypeptide as disclosed herein, which maycomprise spray drying a fermentation medium comprising the polypeptide,or granulating, or encapsulating a polypeptide as disclosed herein, andpreparing the composition.

Nucleic Acids, Expression Vectors, and Recombinant Host Cells

The present disclosure also relates to a nucleic acid which has at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least 99% identity or which has 100% identity to a nucleicacid sequence encoding a polypeptide as disclosed herein. A nucleic acidas disclosed herein may be a nucleic acid which has at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or atleast 99% identity to SEQ ID NO: 2. A nucleic acid as disclosed hereinmay comprise or contain SEQ ID: NO:2. A nucleic acid as disclosed hereinmay further comprise a promotor sequence and/or other control sequence.

A nucleic acid encoding a polypeptide having decolorase activity(including pyropheophytinase activity) as disclosed herein may be acodon optimized, or a codon pair optimized sequence for expression of apolypeptide as disclosed herein in a particular host cell. A host cellmay for instance be Pseudomonas sp, for instance Pseudomonasfluorescens.

In one other embodiment of the present invention a nucleic acid isdisclosed that is an isolated, pure, recombinant, synthetic or variantnucleic acid of the nucleic acid of SEQ ID NO: 2. A variant nucleic acidsequence may for instance have at least 80%, 85% 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequenceidentity to SEQ ID NO: 2.

The present invention also relates to an expression vector comprising anucleic acid as disclosed herein, wherein the nucleic acid is operablylinked to one or more control sequence(s) that direct expression of thepolypeptide in a host cell.

There are several ways of inserting a nucleic acid into a nucleic acidconstruct or an expression vector which are known to a skilled person inthe art, see for instance Sambrook & Russell, Molecular Cloning: ALaboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, N.Y., 2001.It may be desirable to manipulate a nucleic acid encoding a polypeptideof the present invention with control sequences, such as promoter andterminator sequences.

A promoter may be any appropriate promoter sequence suitable for aeukaryotic or prokaryotic host cell, which shows transcriptionalactivity, including mutant, truncated, and hybrid promoters, and may beobtained from polynucleotides encoding extracellular or intracellularpolypeptides either endogenous (native) or heterologous (foreign) to thecell. The promoter may be a constitutive or inducible promoter.Preferably, the promoter is an inducible promoter, for instance a starchinducible promoter.

Promoters suitable in filamentous fungi are promoters which may beselected from the group, which includes but is not limited to promotersobtained from the polynucleotides encoding A. oryzae TAKA amylase,Rhizomucor miehei aspartic proteinase, Aspergillus gpdA promoter, A.niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A.niger or A. awamori glucoamylase (glaA), A. niger or A. awamoriendoxylanase (xlnA) or beta-xylosidase (xlnD), T. reeseicellobiohydrolase I (CBHI), R. miehei lipase, A. oryzae alkalineprotease, A. oryzae triose phosphate isomerase, A. nidulans acetamidase,Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatumDania (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusariumoxysporum trypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the polynucleotides encoding A.niger neutral alpha-amylase and A. oryzae triose phosphate isomerase),and mutant, truncated, and hybrid promoters thereof.

Promoters suitable in bacterial hosts are promoters which may beselected from the group of the E. coli lac promoter, the aroH promoter,the araBAD promoter, the T7 promoter, the trc promoter, the tac promoterand the trp promoter. Other examples of promoters are the promotor ofthe Streptomyces coelicolor agarase gene (dagA), the promoter of theBacillus lentus alkaline protease gene (aprH), the promoter of theBacillus licheniformis alkaline protease gene (subtilisin Carlsberggene), the promoter of the Bacillus subtilis levansucrase gene (sacB),the promoter of the Bacillus subtilis alpha amylase gene (amyE), thepromoter of the Bacillus licheniformis alpha amylase gene (amyL), thepromoter of the Bacillus stearothermophilus maltogenic amylase gene(amyM), or the promoter of the Bacillus amyloliquefaciens alpha-amylasegene (amyQ). Another example is a “consensus” promoter having thesequence TTGACA for the “−35” region and TATAAT for the “−10” region.

The present invention also relates to a recombinant host cell comprisinga nucleic acid as disclosed herein, or an expression vector asdisclosed, wherein the nucleic acid is heterologous to the host cell. Arecombinant host cell as disclosed herein may be a host cell wherein thenucleic acid and the encoding polypeptide having decolorase (includingpyropheophytinase) activity as disclosed herein are heterologous to thehost cell.

A host cell as disclosed herein may be any suitable microbial, plant orinsect cell. A suitable host cell may be a fungal cell, for instancefrom the genus Acremonium, Aspergillus, Chrysosporium, Fusarium,Penicillium, Rasamsonia, Trichoderma, Saccharomyces, Kluyveromyces,Pichia, for instance Aspergillus niger, Aspergillus awamori, Aspergillusfoetidus, A. oryzae, A. sojae, Rasamsonia emersonii Chrysosporiumlucknowense, Fusarium oxysporum, Trichoderma reesei or, Saccharomycescerevisiae, Kluyveromyces lactis, or Pichia pastoris.

A host cell may be a prokaryotic cell, such as a bacterial cell. Theterm “bacterial cell” includes both Gram-negative and Gram-positivemicroorganisms. Suitable bacteria may be from the genus Escherichia,Pseudomonas, Bacillus, Enterobacter, Lactobacillus, Lactococcus, orStreptomyces. A bacterial cell may be from the species B. subtilis, B.amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B.halodurans, B. pumilus, Pseudomonas zeaxanthinifaciens, Pseudomonasfluorescens, or E. coli.

A suitable bacterial host cell may for instance be a Pseudomonas sp.,such as Pseudomonas fluorescens.

Methods of Polypeptide Production

Polypeptides suitable for use in the processes of the present disclosure(e.g, polypeptides having decolorase activity, including a polypeptidehaving pyropheophytinase activity, can be produced by cultivating a hostcell as disclosed herein in a suitable fermentation medium underconditions that allow expression of the polypeptide and producing thepolypeptide. A skilled person in the art understands how to perform aprocess for the production of a polypeptide as disclosed hereindepending on a host cell used, such as pH, temperature and compositionof a fermentation medium. Host cells can be cultivated in shake flasks,or in fermenters having a volume of 0.5 or 1 litre or larger to 10 to100 or more cubic metres. Cultivation may be performed aerobically oranaerobically depending on the requirements of a host cell. In the eventthe host cell is Pseudomonas sp., for instance Pseudomonas fluorescens,cultivation of the host cell is performed under aerobic conditions.

Advantageously, a polypeptide as disclosed herein is recovered orisolated from the fermentation medium, for instance by centrifugation orfiltration known to a person skilled in the art. Recovery of apolypeptide having pyropheophytinase activity may also comprisedisruption of the cells wherein the polypeptide is produced. Disruptionof cells can be performed using glass beads and, or sonification knownto a person skilled in the art.

Processes for Treating Oils Comprising Chlorophyll Derivatives with aPolypeptide Having Decolorase Activity

In one embodiment, the processes of the present disclosure also comprisetreating an oil, comprising a chlorophyll derivative (such apyropheophytin) with a polypeptide having decolorase activity asdisclosed herein, or with a composition comprising a polypeptide asdisclosed herein above. In one embodiment, the polypeptide as disclosedherein has pyropheophytinase activity. In another embodiment, thepolypeptide has pheophytinase activity. In another embodiment, thepolypeptide has pyropheophytinase activity and pheophytinase activity.In another embodiment, the polypeptide has pyropheophytinase activity,pheophytinase activity, and chlorophyllase activity.

As discussed herein, a polypeptide having pyropheophytinase activity iscapable of hydrolyzing pyropheophytin into pyropheophorbide, apolypeptide having pheophytinase activity is capable of hydrolyzing thepheophytin into pheophorbide, and a polypeptide having chlorophyllaseactivity is capable of hydrolyzing chlorophyll into chlorophyllide.Thus, in one embodiment, the decolorase treatment may reduce the levelof one or more chlorophyll substrate in the oil. In various embodiments,the chlorophyll substrate may be chlorophyll, pheophytin, and/orpyropheophytin. For example, the treatment with the decolorase mayreduce the total concentration of chlorophyll substrates in the oil byat least 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, or by 100% by weight, compared to the totalconcentration of chlorophyll substrate (by weight) present in the oilprior to treatment. The reduction in total concentration of chlorophyllsubstrate may be the result of conversion of pyropheophytin intopyropheophorbide, pheophytin into pheophorbide, and/or chlorophyll intochlorophyllide.

In another embodiment, the chlorophyll substrate in the oil comprisespyropheophytin, and at least a portion of the pyropheophytin isconverted into pyropheophorbide as a result of the decolorase treatment.For example, the decolorase treatment may reduce the total concentrationof pyropheophytin in the oil by at least 5%, at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 99%, or by 100% byweight, compared to the total concentration of pyropheophytin (byweight) present in the oil prior to decolorase treatment. The reductionin total concentration of pyropheophytin may be the result of conversionof pyropheophytin into pyropheophorbide. In another embodiment, thechlorophyll substrate in the oil comprises pheophytin, and at least aportion of the pheophytin is converted into pheophorbide as a result ofthe treatment. For example, the decolorase treatment may reduce thetotal concentration of pheophytin in the oil by at least 5%, at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least99%, or by 100% by weight, compared to the total concentration ofpheophytin (by weight) present in the oil prior to decolorase treatment.The reduction in total concentration of pheophytin may be the result ofconversion of pheophytin into pheophorbide. In such embodiments, thepolypeptide exhibits pheophytinase activity.

In another embodiment, the chlorophyll substrate in the oil compriseschlorophyll, and at least a portion of the chlorophyll is converted intochlorophyllide as a result of the decolorase treatment. For example, thedecolorase treatment may reduce the total concentration of chlorophyllin the oil by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 99%, or by 100% by weight, compared tothe total concentration of chlorophyll (by weight) present in the oilprior to decolorase treatment. The reduction in total concentration ofchlorophyll may be the result of conversion of chlorophyll intochlorophyllide. In such embodiments, the polypeptide exhibitschlorophyllase activity.

An oil comprising pyropheophytin, pheophytin, and/or chlorophyll mayfurther comprise other substrates such as phospholipids. Optionally, aprocess for treating an oil as disclosed herein further comprisesremoval of phospholipids, as described hereinafter.

Any oil comprising a chlorophyll derivative, including a chlorophyllsubstrate, may be treated in accordance with the present process inorder to remove one or more undesirable chlorophyll derivative from theoil. The oil may be a triacylglycerol-based oil, including variousvegetable- or algal-based oils. In one embodiment, suitable oils thatmay be used in connection with the present treatment include, but arenot limited to the following: canola oil, castor oil, coconut oil,coriander oil, corn oil, cottonseed oil, hazelnut oil, hempseed oil,linseed oil, mango kernel oil, meadowfoam oil, neat's foot oil, oliveoil, palm oil, palm kernel oil, palm olein, peanut oil, rapeseed oil,rice bran oil, safflower oil, sasanqua oil, sesame oil, soybean oil,sunflower or sunflower seed oil, tall oil, tsubaki oil, vegetable oil,and oil from algae. In one embodiment, an oil that can be treated inaccordance with the present disclosure is selected from the groupconsisting of canola oil, corn oil, olive oil, palm oil, palm kerneloil, peanut oil, rapeseed oil, rice bran oil, sesame oil, soybean oiland sunflower seed oil. In one embodiment, the oil is an oil from algae.

Contacting an oil comprising one or more chlorophyll substrate with apolypeptide having decolorase activity may be performed during anysuitable time and at any suitable pH and temperature. Said contactingmay be performed at a pH and temperature which are applied duringdegumming of a triacylglycerol oil. A suitable pH may be from pH 2 to pH10, for instance from pH 3 to pH 9, from pH 4 to pH 8, from pH 5 to pH7, from pH 5 to 8, or from pH 6.5 to 7.5. In one embodiment, thepolypeptide is contacted with the oil at a pH of from 4.0 to 7.5, orfrom 4.5 to 8.0, or from 4.5 to 7.0. In one embodiment, the polypeptideis contacted with the oil at a pH of from 4.0 to 5.0, or morespecifically at a pH of 4.5. In another embodiment, the polypeptide iscontacted at a pH of 7.0.

A suitable temperature for contacting an oil comprising one or morechlorophyll substrate with a polypeptide having decolorase activity asdisclosed herein may be from 10° C. to 90° C., for instance from 20° C.to 80° C., from 30° C. to 70° C., from 45° C. to 70° C., from 40° C. to60° C., or from 50° C. to 65° C.

For instance, contacting an oil comprising one or more chlorophyllsubstrate with a polypeptide having decolorase activity may be performedat a pH of from 5 to 8, and a temperature of from 40° C. to 60° C., orat a pH of from 4.5 to 7.0 and a temperature of from 40° C. to 60° C.,or at a pH of 7.0 and a temperature of from 45° C. to 70° C., or at a pHof 7.0 and a temperature of from 50° C. to 65° C.

The polypeptide having decolorase activity may be dosed into the oilcomprising a chlorophyll substrate in any suitable amount. For example,the polypeptide may be dosed in a range of 1 to 50 U/gram of treatedoil, such as from 1.4 to 50 U/gram of treated oil, or 5 to 50 U/gram oftreated oil. One unit is defined in accordance with the enzyme activitytaught in the examples below.

Surprisingly, it was found that a polypeptide having decolorase, such aspyropheophytinase activity as disclosed herein converts a higher amountof chlorophyll substrates to chlorophyll products under acidic andcaustic conditions as compared to a reference polypeptide. A referencepolypeptide is a polypeptide comprising the amino acid sequenceaccording to SEQ ID NO: 12. SEQ ID NO: 12 comprises Chlamydomonasreinhardtii chlorophyllase having pyropheophytinase activity.

Contacting an oil comprising one or more chlorophyll substrate with apolypeptide having decolorase activity may be performed during oildegumming. Oil degumming comprises several processing steps, such aspressing and/or hexane extraction, degumming, for instance in thepresence of degumming enzymes such as phospholipases as disclosed inWO2005/086900 or WO2011/046812, refining, bleaching and deodorization.Contacting an oil comprising one or more chlorophyll substrate with apolypeptide having pyropheophytinase activity, pheophytinase activity,and/or chlorophyllase activity may be performed during a bleaching stepin oil degumming processing, as described in more detail hereinafter.

Contacting a polypeptide having pyropheophytinase activity with an oil,such as a triacylglycerol oil or an algal oil, may comprise dispersingan aqueous solution comprising the polypeptide as disclosed herein inthe oil. An oil that is treated with a polypeptide havingpyropheophytinase activity typically comprises 0.5 to 10 w/w % of water,for instance 1 to 10 w/w % of water, 1 to 5 w/w % of water, 2 to 8 w/w %of water, 2 to 4 w/w % of water, 3 to 6 w/w % of water, 0.5 to 5 w/w %of water, 1 to 3 w/w %, 1.5 to 2 w/w % of water, or 5 w/w % water.

The polypeptide may be contacted with the oil for a period of from 5minutes to 24 hours, from 10 minutes to 12 hours, from 15 minutes to 10hours, from 0.5 to 24 hours, from 1 to 12 hours, from 1.5 to 6 hours, orfrom 2 to 4 hours. In one embodiment, the polypeptide may be contactedwith the oil for 2 hours. After said contacting, a water phase and anoil phase are usually separated.

An oil that is treated in a process as disclosed herein may be a crudenon-degummed, degummed (water degummed, enzyme degummed, or aciddegummed), caustic refined or a caustic refined and water washed oil ora water degummed oil. In one particular embodiment, the oil comprises anon-degummed crude oil. A crude oil usually is an oil that ismechanically pressed, or solvent extracted, and wherein the oil usuallycontains Free Fatty Acids (FFA) and phospholipids. A degummed oil is anoil wherein the majority of phospholipids have been removed from a crudeoil. Usually a degummed oil comprises between 0.5 to 200 ppm atomicphosphorous, such as between 1 and 100 ppm atomic phosphorous, such asbetween 5 and 50 ppm atomic phosphorous. A refined oil is an oil wherethe FFA have been neutralized by a caustic treatment and removed. Acaustic treatment of oil usually comprises treating an oil with sodiumhydroxide.

Contacting a polypeptide having decolorase (e.g., pyropheophytinase)activity as disclosed herein with an oil, for instance during degummingof an oil may be performed at any suitable temperature, for instance ata temperature from 45 to 70° C., including from 50 to 65° C.

Contacting a polypeptide having decolorase (e.g., pyropheophytinase)activity as disclosed herein with an oil, for instance during degummingof an oil may be performed at any suitable pH, such as pH of from 3.5 to8.0, for instance pH 4 to 7.5, for instance pH 4.5 to 7.0

In one embodiment, the treatment process disclosed herein furthercomprises subjecting the oil to water degumming. As discussed herein,water degumming is usually applied to crude oils containing a highamount of hydratable phospholipids. Due to its mild characteristics, thephospholipids obtained can be used as lecithin (a natural emulsifier).The oil obtained from this process is generally referred to in theindustry as being “degummed,” despite being only partially degummed.

Thus, in one aspect, the treatment process of the present disclosurecomprises contacting an oil (e.g., a non-degummed crude oil), comprisinga chlorophyll derivative (such as a chlorophyll substrate), with waterand a polypeptide of the present disclosure. Typically, the temperatureof the oil is from 45 to 70° C., including from 50 to 65° C. The watermay be added in an amount of from 1 to 5 w/w %, including from 2 to 4w/w %. The polypeptide may be dosed in an amount of 1 to 50 U/gram oftreated oil, such as from 1.4 to 50 U/gram of treated oil, or 5 to 50U/gram of treated oil. The polypeptide as disclosed herein and water maybe added as a single composition, or the polypeptide may be addedseparately from the water. Typically, no acid or base is added to theresulting mixture, and the process proceeds at a neutral pH (e.g.,around pH 7.0). Following contact with the polypeptide and water, theoil may optionally be mixed using a shear mixer. The oil is subsequentlyincubated with stirring (e.g., using a continuously stirred reactor) forfrom 0.5 to 24 hours, or 1 to 12 hours, or 1.5 to 6 hours, or 2 to 4hours, which aids in hydration of phospholipids present in the oil.Following incubation and stirring, the oil is heated to a temperature offrom 70 to 85° C. The resulting oil may be separated by settling,filtration, or the industrial practice of centrifugation. The centrifugeyields two streams, water degummed oil and wet gums.

In another embodiment, the treatment process disclosed herein furthercomprises subjecting an oil to enzyme assisted water degumming. Asdiscussed herein, enzyme assisted water degumming is usually applied tocrude oils containing a high amount of hydratable phospholipids wherethe goal is to react all of the hydratable phospholipids and convertthem into diacylglycerols increasing the oil yield, while maintainingthe no-hydratable phospholipids in the oil. Enzymes utilized for thisprocess include phospholipase C (PLC) and phosphatidylinositol-phospholipase (PI-PLC).

Thus, in one aspect, the treatment process of the present disclosurecomprises contacting an oil (e.g., a non-degummed crude oil), comprisinga chlorophyll derivative (such as a chlorophyll substrate) with water, apolypeptide of the present disclosure, and an additional enzyme. Theadditional enzyme may be selected from the group consisting of PLC,PI-PLC and combinations thereof. In one embodiment, the additionalenzyme includes both PLC and PI-PLC. Typically, the temperature of theoil is from 45 to 70° C., including from 50 to 65° C. The water may beadded in an amount of from 1 to 5 w/w %, including from 2 to 4 w/w %.The polypeptide may be dosed in an amount of 1 to 50 U/gram of treatedoil, such as from 1.4 to 50 U/gram of treated oil, or 5 to 50 U/gram oftreated oil. The PLC (e.g., Purifine PLC) may be added in an amount offrom 50 to 500 ppm, including from 100 to 400 ppm, or from 150 to 250ppm. The PI-PLC may be added in an amount of from 50 to 500 ppm,including from 100 to 400 ppm, or from 150 to 250 ppm. In oneembodiment, the additional enzyme is Purifine 4G, which contains bothPLC and PI-PLC. In this embodiment, the Purifine 4G may be added in anamount of from 50 to 500 ppm, including from 100 to 400 ppm, or from 150to 250 ppm. The polypeptide as disclosed herein, additional enzymes, andwater may be added as a single composition, or the polypeptide asdisclosed herein, additional enzymes, and water may be added separately.Typically, no acid or base is added to the resulting mixture, and theprocess proceeds at a neutral pH (e.g., around pH 7.0). Followingcontact with the polypeptide, additional enzymes, and water, thecomposition may optionally be mixed using a shear mixer. A suitableshear mixer is the continuous shear mixer IKA Dispax Reactor. Thecomposition is subsequently incubated with stirring (e.g., using acontinuously stirred reactor) for from 0.5 to 24 hours, or 1 to 12hours, or 1.5 to 6 hours, or 2 to 4 hours, which aids in conversion ofPC, PE, and PI to diacylglycerols in the oil. Following incubation andstirring, the composition is heated to a temperature of from 70 to 85°C., such as 85° C. The resulting composition may be separated bysettling, filtration, or the industrial practice of centrifugation. Thecentrifuge yields two streams, water degummed oil and heavy phase(containing water, denatured protein, and phosphor-compounds).

In another embodiment, the treatment process disclosed herein furthercomprises subjecting the oil to enzyme degumming. Enzyme degumming maybe applied to crude oils or to oils that have been degummed previouslyby a different method, such as water degumming, enzyme assisted waterdegumming, or acid degumming. A processor who wishes to produce lecithinfor the food or industrial market may water degum the oil prior tofurther processing. The destruction of the phospholipids is unacceptablein lecithin applications.

Thus, in one aspect, the treatment process of the present disclosurecomprises contacting a composition, such as an oil (e.g., a crude oil orpreviously degummed oil), comprising a chlorophyll derivative (such as achlorophyll substrate), with a polypeptide of the present disclosure.The pH of the oil may be adjusted prior to contacting with thepolypeptide, for example by addition of an acid (e.g., citric orphosphoric acid) in an amount of from 100 to 1000 ppm, including 500ppm. Typically, the pH is adjusted to a pH of from 4.5 to 8.0, includingfrom 4.5 to 7.0. Typically, the temperature of the oil is from 70 to 85°C. at the time of pH adjustment. Following acid addition, the resultingoil may be mixed for from 5 minutes to 24 hours, depending on the typeof mixer (e.g., high shear, agitator, etc.). One skilled in the art willunderstand that lower mixing times will be needed when high shear mixersare used, while higher mixing times will be needed when less shear isapplied (e.g., when using a simple agitator). Following pH adjustment,the composition (e.g., oil) is cooled to from 45 to 70° C., includingfrom 50 to 65° C., and water, a polypeptide of the present disclosure,and optionally an additional enzyme are added. The additional enzyme(when used) may be selected from the group consisting of PLC, PI-PLC,and combinations thereof. In one embodiment, the additional enzymeincludes both PLC and PI-PLC. The water may be added in an amount offrom 1 to 5 w/w %, including from 2 to 4 w/w %. The polypeptide may bedosed in an amount of 1 to 50 U/gram of treated oil, such as from 1.4 to50 U/gram of treated oil, or 5 to 50 U/gram of treated oil. The PLC(e.g., Purifine PLC) may be added in an amount of from 50 to 500 ppm,including from 100 to 400 ppm, or from 150 to 250 ppm. The PI-PLC may beadded in an amount of from 50 to 500 ppm, including from 100 to 400 ppm,or from 150 to 250 ppm. In one embodiment, the additional enzyme isPurifine 4G, which contains both PLC and PI-PLC. In this embodiment, thePurifine 4G may be added in an amount of from 50 to 500 ppm, includingfrom 100 to 400 ppm, or from 150 to 250 ppm. The polypeptide, additionalenzyme (when present), and water may be added as a single composition,or the polypeptide, additional enzymes, and water may be addedseparately.

Following contact with the polypeptide, additional enzyme (whenpresent), and water, the composition may be mixed using a shear mixer. Asuitable shear mixer is the continuous shear mixer IKA Dispax Reactor.Shear mixing is optional, particularly when the composition beingtreated is, or comprises, a crude, non-degummed oil. The composition issubsequently stirred (e.g., using a continuously stirred reactor) forfrom 0.5 to 24 hours, or 1 to 12 hours, or 1.5 to 6 hours, or 2 to 4hours.

Following stirring, a phospholipase A (PLA) enzyme is added to the oil.The PLA enzyme may be a PLA1 enzyme and/or PLA2 enzyme. In oneembodiment, the enzyme is a PLA1 enzyme. Sequences of amino acids withphospholipase activity are extensively reported in the art, includingphospholipids having activity in triacylglycerol oils. Commercial PLA1enzymes with phospholipase activity include Lecitase® Ultra andQuaraLowP. Commercial PLA2 enzymes with phospholipase activity includeRohalase Xtra and LysoMax. Any suitable PLA enzyme may be PLA added mayvary depending on the manufacturer and the type of continuousstirred-tank reactor used. Following addition of the PLA enzyme, the oilmay be mixed using a shear mixer. A suitable shear mixer is thecontinuous shear mixer IKA Dispax Reactor. The oil is subsequentlyincubated with stirring (e.g., using a continuously stirred reactor).The oil may be incubated with the PLA1 enzyme allowed to react for from1 to 8 hours, or 2 to 7 hours, or 3 to 6 hours. Following incubation andstirring, the oil is heated to a temperature of from 70 to 85° C., suchas 85° C. Reaction times may vary, depending on the PLA dosage and thelevel of non-hydratable phospholipids (NHPs) (e.g., Ca and Mg salts ofphosphatidic acid) present. The resulting oil may be separated bysettling, filtration, or the industrial practice of centrifugation.

In another embodiment, the treatment process of the present disclosurecomprises contacting an oil, in particular a once refined oil,comprising a chlorophyll derivative (such as a chlorophyll substrate),with a polypeptide of the present disclosure. Typically, the temperatureof the oil is from 45 to 70° C., including from 50 to 65° C. Thepolypeptide may be dosed in an amount of 1 to 50 U/gram of treated oil,such as from 1.4 to 50 U/gram of treated oil, or 5 to 50 U/gram oftreated oil. Water may be added to the oil in an amount of from 1 to 10w/w %, including from 2 to 8 w/w %, or 3 to 6 w/w %, or 5 w/w %. Thepolypeptide and water may be added as a single composition, or thepolypeptide may be added separately from the water. Typically, no acidor base is added to the resulting mixture, and the process proceeds at aneutral pH (e.g., pH 7.0). Following contact with the polypeptide andwater, the oil may be mixed using a shear mixer. A suitable shear mixeris the continuous shear mixer IKA Dispax Reactor. The oil is incubatedwith stirring for from 1.5 to 3 hours, including 2 hours. Followingincubation and stirring, the oil is heated to a temperature of from 70to 85° C. The resulting oil may be separated by settling, filtration, orthe industrial practice of centrifugation.

In another embodiment a process for treating an oil comprising achlorophyll derivative as disclosed herein may further comprise removalof pyropheophorbide, and/or pheophorbide. In one embodiment, a processfor treating an oil comprising a chlorophyll derivative as disclosedherein may further comprise removal of chlorophyllide, pyropheophorbide,and/or pheophorbide. Pyropheophorbide, pheophorbide, and/orchlorophyllide can be removed during a water wash of the oil, during achemical refining step (addition of water to remove excess soap), or byusing an adsorbent of the present disclosure in the deodorization step.

In one embodiment, a process for treating an oil comprising achlorophyll derivative, such a pyropheophytin, may further comprisetreating the oil with an additional enzyme selected from the groupconsisting of a phospholipase, a chlorophyllase, a pheophytinase, apyropheophytinase, and combinations thereof. A suitable phospholipasemay be a phospholipase A, phospholipase B and/or phospholipase C or anysuitable combination of these enzymes. Treating the oil with aphospholipase, so-called enzymatic degumming, reduces the phospholipidcontent in the oil, resulting in a lower atomic phosphorous content inthe oil.

The present disclosure also relates to an oil (e.g, a triacylglyceroloil, vegetable oil, oil from algae, etc.) obtainable by a process asdisclosed herein. An oil, which may be a triacylglycerol oil obtainableby a process as disclosed herein may comprise a polypeptide havingdecolorase activity, such as pyropheophytinase activity as disclosedherein.

FIGURES

FIG. 1 : Overview of the conversion of chlorophyll into pheophytin andpyropheophytin and into the respective reaction products chlorophyllide,pheophorbide and pyropheophorbide. The A compounds are shown, which havea methyl group at the C7 position. B compounds have an aldehyde in theC7 group instead of a methyl group. Structures are taken from PubChem,NCBI.

FIGS. 2-2A: HPLC results of incubation pheophytin a and b andpyropheophytin a and b with different putative chlorophyllases at pH 7and 50° C., for 24 hours. The amounts of the substrates pheophytin a andb and pyropheophytin a and b and the reaction products pheophorbide aand b and pyropheophorbide a and b are given as peak surface areas. Thefirst two columns show the sum of reaction products and substrates. “nd”means: not detectable.

FIG. 3 : HPLC results of incubation pheophytin a and b andpyropheophytin a and b with different putative chlorophyllases at pH 5and 50° C., for 24 hours. The amounts of the substrates pheophytin a andb and pyropheophytin a and b and the reaction products pheophorbide aand b and pyropheophorbide a and b are given as peak surface areas. Thefirst two columns show the sum of reaction products and substrates. “nd”means: not detectable.

FIG. 4 : 4A: Chlorophyll derivatives 4B: Phosphor compounds in canolaoil after 24 h incubation with CHL26 enzyme from Hordeum vulgare or thereference enzyme ELDC94 from Chlamydomonas reinhardtii.

FIG. 5 : 5A: Chlorophyll derivatives, and 5B: Phosphor compounds incanola oil and soy bean after several incubations with CHL26 enzyme fromHordeum vulgare and/or the reference enzyme ELDC94 from Chlamydomonasreinhardtii, under different reaction conditions and 5C: chlorophyllderivatives in the obtained gums.

FIGS. 6-6A: Chlorophyll derivatives in canola oil and soybean oil aftercaustic refining and after incubation with CHL26 enzyme from Hordeumvulgare or the reference enzyme ELDC94 from Chlamydomonas reinhardtii.

FIG. 7 : Schematic presentation of a typical chemical refinery processfor triacylglycerol based oils. A process of solvent extraction and/orpressing on an oilseed (rapeseed or soybean), oil fruit plant (palm), orsingle cell source (algal) to obtain a crude oil. The crude oil thisthen treated with citric or phosphoric acid to react with thenon-hydratable phospholipids and then the addition of sodium hydroxideto neutralize the free fatty acids and form sodium soaps. The sodiumsoaps form an emulsion with the water present allowing the removal ofnon-hydratable phospholipids when the oil is centrifuged to producerefined oil. The refined oil may then be washed with hot water andcentrifuged to remove the remaining soaps and phospholipids.Alternatively, the refined oil may be treated with acidic silica toadsorb soaps, trace metals and phospholipids. The industrial acidicsilicas do not have any capacity to remove chlorophyll or chlorophyllderivatives. The oil is then treated with bleaching earth to remove thesoaps, phospholipids, and chlorophyll and chlorophyll derivativespresent in the oil. The final step in the deodorization step of steamdistillation at elevated temperatures and vacuums of less than 5 mBar.The distillation primarily removes peroxides, aldehydes, ketones andother flavor compounds. It also destroys beta-carotene and removes theremaining free fatty acids (0.1 percent) to reach a level of 0.02 to0.05% final Free Fatty Acid (FFA).

FIG. 8 : Schematic presentation of a typical enzymaticdegumming/physical refining process. The crude oil is treated withphosphoric or citric acid to enable the non-hydratable phospholipids tolose the calcium or magnesium bond to them at a pH of roughly 2. Thesodium hydroxide is then added to bring the pH above 4 for citric acidor above 6 for phosphoric acid in order that the phospholipase may workand obtain a very low residual phosphorus<5 ppm) after the enzymaticreaction with the PLAs. Alternatively, the PLAs may be reacted with thePLC and/or PI-PLC to maximize the oil yield and still obtain a very lowresidual phosphorus allowing for physical refining. The oil is theneither washed or treated with an acidic silica followed or incombination with bleaching earth. After the bleaching process withchlorophyll levels of less than 50 ppb, the oil is physically refined inthe deodorizer. The high temperature steam distillation removes all ofthe compounds describe above in FIG. 7 , but its primary purpose is theremoval of FFA. The FFAs are distilled and collected in the scrubber.Very limited neutral oil is lost in the deodorization process comparedto the losses associated from the emulsions formed in water degumming orchemical refining.

FIG. 9 : Schematic presentation of the use of a decolorase enzyme in thewater degumming process or the enzyme assisted water degumming process.A decolorase enzyme may be added with the water at 60° C., or with thePLC, or with the combination of PLC and PI-PLC. After two hours ofincubation, the oil is heated to 70 to 85° C. and centrifuged to removethe reacted gums and reacted chlorophyll derivatives.

FIG. 10 : Schematic presentation of an enzymatic degumming processmodified to include treatment with a decolorase enzyme and a silicaadsorbent (such as a MgO-treated adsorbent) of the present disclosure.The crude oil is first treated with citric acid to a pH of roughly 2 todissociate the bond calcium and magnesium ions, the pH is raised above 4to enable the PLCs and Decolorase enzymes in a pH that enable them towork efficiently. 1 to 5 percent water is added for the hydrolysisreactions. After the completion of the PLCs and Decolorase incubations,a PLA1 or PLA2 may be added to react with the non-hydratablephospholipids present in the oil. After an additional incubation of 2 to6 hours, the oil is heated to 70 to 85° C. and centrifuged to remove thereacted gums and chlorophyll derivatives producing an oil with less than5 ppm residual phosphorus in the oil. The enzymatically degummed oil maythen be contacted with a silica adsorbent of the present disclosure,optionally under vacuum, to further remove chlorophyll derivatives andtrace metals, as described herein.

FIG. 11 : Schematic presentation of a chemical refining process with adecolorase enzyme, followed by treatment with a silica adsorbent of thepresent disclosure. The decolorase enzyme may not be added in the acidor caustic addition steps due to the very low pH (roughly 2) and thevery high pH (roughly 14) in the early steps of the process. Thedecolorase enzyme must be added after the initial centrifuge step in therefined oil. It is advantageous to add the decolorase enzyme with thewashing step at a temperature suitable for the enzyme (50 to 65° C.).Allow an incubation time of at least two hours followed by heating to 70to 85° C. prior to centrifugation. The oil would then be furtherprocessed by contacting with a silica adsorbent of the presentdisclosure, optionally under vacuum, to further remove chlorophyllderivatives and/or trace metals, as described herein.

FIG. 12 : Schematic presentation of an enzymatic degumming/physicalrefining process modified to include treatment with a silica adsorbentof the present disclosure, but without decolorase treatment. Theenzymatically degummed oil or the enzymatically degummed and washed oilis contacted with a silica adsorbent of the present disclosure, andoptionally with bleaching earth, optionally under vacuum, to removechlorophyll derivatives and trace metals, as described herein.

FIG. 13 : Schematic presentation of a chemical refinery process fortriacylglycerol based oils, modified to include treatment with a silicaadsorbent of the present disclosure, but without decolorase treatment.The refined oil or the once refined oil is contacted with a silica-basedadsorbent of the present disclosure, and optionally with bleachingearth, optionally under vacuum, to remove chlorophyll derivatives andtrace metals, as described herein.

SEQUENCES

SEQ ID NO: 1=CHL26 polypeptide having decolorase including apyropheophytinase activity from Hordeum vulgare.

MASAGDVFDHGRHGTSLARVEQAKNTRCSAASRVDADAQAQQSPPKPLLVAAPCDAGEYPVVVFLHGYLCNNYFYSQLIQHVASHGFIVVCPQLYTVSGPDTTSEINSAAAVIDWLAAGLSSKLAPGIRPNLAAVSISGHSRGGKVAFALGLGHAKTSLPLAALIAVDPVDGTGMGNQTPPPILAYKPNAIRVPAPVMVIGTGLGELPRNALFPPCAPLGVSHAAFYDECAAPACHLVARDYGHTDMMDDVITGAKGLATRALCKSGGARAPMRRFVAGAMVAFLNKWVEGKPEWLDAVREQTVAAPVVLSAVEFRDE

SEQ ID NO: 2: Codon optimized nucleic acid sequence encoding apolypeptide having decolorase including pyropheophytinase activity fromHordeum vulgare CHL26 for expression in Pseudomonas fluorescens.

SEQ ID NO: 3; CHL25 putative chlorophyllase from Gossypium raimondii

SEQ ID NO: 4; CHL27 putative chlorophyllase from Phoenix dactylifera

SEQ ID NO: 5; CHL28 putative chlorophyllase from Wollemia nobilis

SEQ ID NO: 6; CHL29 putative chlorophyllase from Cucumis sativus

SEQ ID NO: 7; CHL30 putative chlorophyllase from Tarenaya hassleriana

SEQ ID NO: 8; CHL31 putative chlorophyllase from Solanum tuberosum

SEQ ID NO: 9; CHL32 putative chlorophyllase from Populus trichocarpa

SEQ ID NO: 10; CHL33 putative chlorophyllase from Vigna radiata

SEQ ID NO: 11; N1 Negative control, Green Fluoresent Protein (GFP)

SEQ ID NO: 12; P2, Chlamydomonas reinhardtii chlorophyllase havingpyropheophytinase activity. SEQ ID NO: 12 is also referred to herein asELDC94.

SEQ ID NO: 13; SpeI site and ribosome binding site

SEQ ID NO: 14; stop codon and XhoI site.

EXAMPLES Materials and Methods General

Standard genetic techniques, such as overexpression of enzymes in thehost cells, genetic modification of host cells, or hybridisationtechniques, are known methods in the art, such as described in Sambrookand Russel (2001) “Molecular Cloning: A Laboratory Manual (3^(rd)edition), Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, or F. Ausubel et al, eds., “Current protocols in molecularbiology”, Green Publishing and Wiley Interscience, New York (1987).Water is Milli-Q water where nothing else is specified.

Analytical Methods:

pH—stoichiometric addition of acid and base to a water percentage thatwas added to the oil.

2 percent water in a 2000 grams reaction would be 40 grams, adding 2.0grams of a 50 percent solution of citric acid, plus 1.6 mL of 4 M sodiumhydroxide would yield a water solution with a pH of 4.5. The pH of theoil will always remain 7.

Soap—American Oil Chemists' Society Official Method Cc 13a-43, revised2017. Free Fatty Acid—American Oil Chemists' Society Official Method Ca5a-40, revised 2017.

Color—American Oil Chemists' Society Official Method Ce 13e-92,reapproved 2017. Utilized Tintometer's PFX-950 at 5¼″ cell.

Phosphorus and trace metals—American Oil Chemists' Society OfficialMethod Ca 17-01-43, revised 2017.

Phospholipid Compositions For ³¹P NMR methods (also referred to as 31-PNMR), 10 μL of 10% DOL dispersion was dispersed in 1 mL of an aqueoussolvent containing demineralized water with 10% deuterium oxide (D₂O,Cambridge Isotope Laboratories, DLM-4), 25 mg/mL deoxycholic acid (SigmaD2510), 5.84 mg/mL EDTA di Na (Titriplex III, Merck 108418), and 5.45mg/mL TRIS base (Tris(hydroxymethyl) aminomethane, Merck 108387), ofwhich the pH was adjusted to pH 9 using 4N KOH and to which 2 mg/mL TIPinternal standard (tri-isopropylphosphate, Aldrich 554669) (accuratelyweighed) was added.

All samples were measured in a Bruker 400 MHz AvanceIII NMR spectrometerwith a Prodigy BBO probe. The temperature of the probe head was set at300K.

The measurement for quantification was performed with semi-quantitativeparameters: 128 scans, 90° pulse, D1=5 sec. Values are reported inμmol/g of dry weight (DOL) of the sample.

Analysis of green color content by UV/Vis—The AOCS UV/Vis method is usedto measure the green color content of oils. The AOCS UV/Vis method isdescribed in Cc 13d-55, reapproved 2017.

Analysis of Chlorophyll Derivatives by HPLC-FLU

The analysis of pheophytins A and B, and pyropheophytins A and B, andtheir phorbides, as well as chlorophyll and chlorophyllide, wasperformed by HPLC using fluorescence detection, a method developed basedon the work of Hwang et al J. Food Hyg. Soc. Japan Vol. 46, No. 2,45-48, extended by fluorescence detection at λex 410 nm/λem 666 nm forthe A compounds, and λex 436 nm/λem 653 nm for the B compounds.

Analysis for the Water Content of the Adsorbent

Water content, in wt %, is determined by heating the adsorbent to 1750 Funtil a constant weight is observed. The water content equals the masslost divided by the original mass of the material expressed inpercentage.

Sample Preparation

Oil samples were diluted in acetone, 1 g oil in 9 mL acetone, andcentrifuged at 14000 rpm for 5 minutes. The clear supernatants weretransferred into injection vials, and 10 μl of a sample was injectedinto the HPLC. As the chlorophyll levels were so low in all practicaloil samples, these were not taken into account in the analysis.

Data Analysis

The peak surface areas (in arbitrary units) of the chromatogramsindicate the amount of pheophytins, pyropheophytins, pheophorbides andpyropheophorbides present in the oil samples. FIGS. 2 and 3 show thepeak surface areas of pheophytins, pyropheophytins, pheophorbides andpyropheophorbides in oil samples after incubation with putativechlorophyllases at pH 5 and pH 7. The sum of the peak surface area ofphytines, the sum of peak surface area of phorbides and the peak surfacearea of the individual compounds are shown. The formation ofpheophorbide and pyropheophorbide is a measure for the presence ofpheophytinase activity and pyropheophytinase activity, respectively.

Enzymes

Purifine® Phospholipase C (PLC), and Purifine® PI-PLC and a fungal PLA1were obtained from DSM.

Purifine® Phospholipase C comprises amino acids 38-282 of SEQ ID NO: 2,having the amino acid substitutions 63D, 131S and 134D disclosed inWO2005/086900

Purifine® PI-PLC comprises the mature polypeptide according to SEQ IDNO: 8 disclosed in WO2011/046812.

Fungal PLA1 comprises the mature amino acid sequence of SEQ ID NO: 1disclosed in European application no. EP18171015.3

Equipment

The overhead mixer was an IKA RW 20 Digital with a flat blade paddle.

The centrifuge was a De Laval Gyro—Tester installed with “The Bowl Unit”for continuous separation. The centrifuge bowl was closed with the plugscrews installed. Shear mixing was accomplished with an Ultra-Turraxhomogenizer SD-45 with a G450 rotor stator at 10,000 rpm.

Silica Adsorbents

Test adsorbents SP-2113, SP-2114, SP-2115, SP-2116, SP-2117, and SP-2119were obtained from W.R. Grace & Co.-Conn. (Columbia, Md.). TRISYL®silica and TRISYL® 300 (W.R. Grace & Co.-Conn., Columbia, Md.) arecommercially available. The properties of various silicas and adsorbentsused in the Examples are set forth below.

Na₂O MgO (wt % (wt % Median Base on a on a Water Particle Silica dry dryContent Size Type pH basis) basis) (wt %) (μm) SP-2113 Hydrogel 10.25.81 <0.1 51.9 20 SP-2114 Hydrogel 8.6 <0.1 5.0 60.2 20.1 SP-2115Hydrogel 8.7 <0.1 11.7 58.4 20.1 SP-2116 Acidic 6.1 2.91 <0.1 53.7 21hydrogel SP-2117 Acidic 6.1 4.88 <0.1 54.6 21 hydrogel SP-2119 Xerogel9.1 0.10 5.2 11.7 19.1 TriSyl ® Acidic 4.5 <0.1 <0.1 60.0 20.0 silicahydrogel TriSyl ® Acidic 2.5 <0.1 <0.1 60.0 20.0 300 hydrogel AbsorbentA Hydrogel 8.9 <0.1 11.7 5.2 — Absorbent B Hydrogel 9.8 <0.1 34.5 48.914.3 Absorbent C Xerogel 8.7 0.10 9.4 51.8 19.4 Absorbent D Xerogel 8.3<0.1 14.7 56.7 151

Example 1 Expression of a Putative Chlorophyllases in Pseudomonas

Putative chlorophyllases (CHL) as provided in the tables of FIGS. 2 and3 were expressed in the Pseudomonas system obtained from Dow GlobalTechnologies Inc. (US20050130160, US20050186666 and US20060110747). The12 synthetic genes based on the protein sequence of the putativechlorophyllases protein sequences as shown in FIG. 2-2A and 3 weredesigned by optimizing the gene codon usage for Pseudomonas according tothe algorithm of DNA2.0 (GeneGPS® technology). For cloning purposes, theDNA sequence contain a SpeI site and ribosome binding site(ACTAGTAGGAGGTAACTAATG) (SEQ ID NO: 13) at the 5′-end and a stop codonand XhoI site (TGATGACTCGAG) (SEQ ID NO: 14) at the 3′-end.

SEQ ID NO: 2 shows the codon optimized nucleic acid sequence encodingthe putative chlorophyllase SEQ ID NO:1 of Hordeum vulgare.

The DNA sequences were inserted in the pDOW1169 vector (Dow GlobalTechnologies Inc., US20080058262) using SpeI and XhoI restriction enzymecloning. The pDOW1169 vectors containing the genes encoding the CHL andPPH enzymes under control of a modified tac promotor were thenintroduced into Pseudomonas fluorescens uracil auxotrophic strain DPfl0.The transformed cells were selected after incubating on M9 minimalmedium at 30° C. for 48 hours (Dow Global Technologies Inc.,US20050186666) without uracil (Schneider et al. 2005).

Correct transformants were pre-cultured in 24 well pre-sterile deep wellplates (Axygen, Calif., USA) containing 3 ml M9 medium. Plates werecovered by a Breathseal (Greiner bio-one, Frickenhausen, Germany) andincubated at 30° C., 550 rpm and 80% humidity for 16 hours in a Microtonincubator shaker (Infors AG, Bottmingen, Switzerland). From thesecultures 30 μl was used to inoculate a second 24 well pre-sterile deepwell plates (Axygen, Calif., USA) containing 3 ml M9 medium at 30° C.,550 rpm for 24 hours. After 8 hours, the cultures were induced with IPTG(0.3 mM final concentration). Cultures were harvested by centrifugationfor 10 minutes at 2750 rpm and the supernatants removed. The cellpellets were stored overnight at −20° C. The cell pellets from the 3 mlcultures were suspended in 1 ml lysis buffer and incubated for one hourat 37° C. Lysis buffer (1 mM EDTA, 50 mM Tris, pH 8, 0.25 mg/mllysozyme, 10 mg/ml DnaseI, 25 μM MgSO₄ and 0.03% triton). The lysateswere centrifuged at 2750 rpm for 10 minutes and the supernatants wereremoved and stored.

Example 2 Determination of Pyropheophytinase Activity in Cell-FreeExtracts in Crude Canola Oil

Incubation

Crude canola oil from North American origin, high in pheophytins andpyropheophytins was used to determine activity of the enzyme in thesupernatant as produced in Example 1 on pyropheophytin A and B in thefollowing way. Buffer (5% (v/v)) was added to oil under high-shearmixing using a Silverson mixer. For pH 5, a 20 mM citric acid buffer wasused. For pH 7 a 20 mM phosphate buffer was used. A 24 wells microtiterplate was filled with 1.425 mL buffer-in-oil dispersion per well, and toeach well 75 μL, 5% (v/v) cell-free extract (supernatant) produced inExample 1 was added. A list of tested samples is given in the tables ofFIG. 2-2A and FIG. 3 , and include a positive reference containingChlamydomonas reinhardtii pyropheophytinase and negative control GreenFluorescent Protein (GFP). The microtiter plate was covered with plasticfoil [Fasson S695]. Each well was stirred with an individual magneticstirring bar. Incubations were performed at 50° C. using a KBMDmicrotiter-plate stirrer. Samples were taken after 24 hours and analysedfor the presence of pheophytins A and B, and pyropheophytins A and B,and their phorbides using HPLC-FLU as described above.

The results in FIGS. 2 and 3 show that only CHL26, a putativechlorophyllase from Hordeum vulgare, was able to hydrolyse allpheophytins and pyropheophytins into their respective(pyro)pheophorbides at pH 7 and pH 5.

Example 3 Incubation of Crude Canola Oil with CHL26 Versus Time

Incubation of crude canola oil with 5% cell free extract of Hordeumvulgare putative chlorophyllase CHL26 produced as described in Example1, was repeated in the same way as described in Example 2 at pH7.Samples were taken after 30 min, 2 hr, 5 hr, and 24 hr. Pyropheophytin aand b, and pheophytin a and b, pyropheophorbide a and b and pheophorbidea and b were measured by HPLC as described above.

The formation of the reaction products pyropheophorbide a and b andpheophorbide a and b in Table 1 is expressed as percentage of the amountreaction product (respective phorbide molecule) after 24 hr.

Table 2 shows the relative amounts of pheophytins and pyropheophytins asa function of time after 0.5, 2 and 5 hr, expressed in percentagesrelatively to the value at t=0 (average of 4 measurements).

TABLE 1 Relative HPLC results for all reaction products after incubationfor 0.5, 2, 5 and 24 hours at pH 7 and 50° C., in percentages relativeto value after 24 hrs. Pheophor- Pyropheophorbide PheophorbidePyropheophorbide time bide B B A A [hr] (%) (%) (%) (%) 0.5 53.7 48.869.4 56.7 2 85.3 95.5 96.4 90.0 5 92.9 92.9 98.7 94.3 24 100.0 100.0100.0 100.0

TABLE 2 Relative HPLC results for all phytin compounds after incubationfor 0.5, 2, and 5 hours at pH 7 and 50° C., in percentages relative tovalue at t = 0. Pheophytin Pyropheophytin Pheophytin Pyropheophytin SumTime B B A A phytins [hr] (%) (%) (%) (%) (%) 0 100.0 100.0 100.0 100.0100.0 0.5 34.7 43.8 34.4 46.4 41.4 2 0.0 12.7 0.0 12.9 8.2 5 0.0 6.2 0.06.0 3.9

The results in Table 1 and 2 show that enzyme CHL26 from Hordeum vulgareis able to hydrolyse both pheophytin and pyropheophytin, and both the aand b compounds. After 2 hrs all pheophytins were converted (belowdetection limit), whereas after 5 hours almost all the pyropheophytinswere converted.

Example 4 Production of CHL26 and ELDC94 by 10 L Bioreactor Fermentation

Strains and Inoculum

Of a P. fluorescens strain containing CHL26 (SEQ ID NO: 1) andChlamydomonas reinhardtii (ELDC94; SEQ ID NO: 12) chlorophyllase asdescribed in Example 1 a pre-culture was prepared in one-phase shakeflasks with complex medium comprising yeast extract, slats and glycerolas a C-source, which was used as inoculum for the 10 L fermentationswith inoculation ratio of 10% described below.

10 L Fermentations

Fermentation process was based on industrial Pseudomonas fluorescensfermentations (fed-batch process, sugar limited, IPTG induced). Thefermentation process consisted of biomass production under exponentialfeed of glucose as C-source followed by production phase under IPTGinduction system. After 23 hr fermentation (end of biomass productionphase), IPTG was added to a final concentration of 0.125 mM in order toinduce enzyme production. The feed rate of C-source (glucose) wasreduced to ˜70% of maximum and fermentation prolonged till 48-55 hoursafter inoculation.

At the end of fermentation, the broth was killed off and the enzymerelease via benzoate treatment followed by pH increase of thefermentation broth.

Recovery

The intra-cellular enzyme was released by homogenization. Two passes at750 bars, with a cooling period of 12-hours in-between was applied.Subsequently the homogenized broth was diluted with 30% water, 15% DBF(Dicalite BF), Calcium Chloride (20 g/kg original broth), and FlocculentC577 (0.1% on original broth) were added. The pH was adjusted to 8, andthe material was clarified and ultra-filtrated. The UF was stabilizedwith 50% glycerol, and to ensure full killing of remaining bacteria MEP(methyl/ethyl paraben in a solution with propene-diol) was added,diluting the product with about 15% v/v.

Activity

Activity on p-NP Substrates

The enzyme activity was determined using the chromogenic substrate4-nitrophenyl butyrate (Sigma N9874). Substrate stock solution: 50 mMpNP-butyrate in acetonitrile. Substrate solution: Prior to use thesubstrate stock solution was mixed in ratio 1:4 with 0.1 M phosphatebuffer pH 7.0 also containing 0.2% BSA and 2.0% Triton X-100.

In micro titer plates, 120 μL phosphate buffer (same as above) was mixedwith 15 μL substrate solution and equilibrated at 37° C. After startingthe reaction by adding 15 μL sample, the OD at 405 nm was measured for 5minutes. Also, a blank measurement was done by adding 15 μL bufferinstead of sample. The slope of the linear part of the curve is used asmeasure for the activity. Samples were diluted such to assure that theabsorbance increase after 5 minutes is less than 1.0.

Activity is calculated as follows:

U/mL=(ΔAbs/min sample−ΔAbs/min blank)/(ε_(pNP)×5)×1000×150/15×Df/W

ε_(pNP)=Molar Extinction Coefficient of para-nitro-phenol [L·mol-1·cm-1]

5=Incubation time [min]

1000=factor from mmol to μmol

150=assay volume [μL]

15=sample volume [μL]

Df=Dilution factor

W=weight of sample (g)

The activity is expressed as the amount of enzyme that liberates 1micromol p-nitrophenol per minute under the conditions of the test.Calibration is done using a 4-nitrophenol standard solution (SigmaN7660) diluted in the above-mentioned phosphate buffer.

The activity of the final formulations of CHL26 was 1.4 U/g (0.5 w/w %),and of ELDC94 87 U/g (0.04 w/w %).

Example 5 Incubation of Crude Canola Oil with an Enzyme HavingPyropheophytinase Activity Derived from Hordeum vulgare (CHL26) Comparedto Incubation of Crude Canola Oil with a Reference Enzyme (ELDC94) fromChlamydomonas reinhardtii at Various Conditions

Crude canola oil was incubated with 0.5 w/w % cell free extract ofHordeum vulgare putative chlorophyllase CHL26 and compared to 0.04 w/w %of cell-free extract of Chlamydomonas reinhardtii chlorophyllase (codedELDC94=Ref) both enzymes produced as described in Example 4. Theincubation was performed on 10 g scale (10 g oil in 15 ml glass reactionvessels incubated on a hot plate aluminium reaction block withtemperature control. Contents are kept vigorously stirred by magneticbars), and now at three different temperatures (40, 50 and 60° C.), andunder four regimes with varying acidity of the aqueous phase:

Acidic: 400 ppm citric acid pre-treatment;

Mildly acidic: pre-treatment with 500 ppm citric acid and 138 ppmcaustic (NaOH);

Neutral: only water;

Mildly alkaline: pre-treatment with 150 ppm NaOH.

The total water level during incubation is 3% w/w, which includes enzymeformulation and pre-treatment solutions. Prior to the experiment, theacidity of the aqueous environment was assessed by diluting thepre-treated oil 1:1 with water and then the pH was measured by a pHmeter. This resulted in the following pH values, indicative for theacidity of the aqueous environment in the dispersion during reaction:Acidic: pH 3.4; mildly acidic: pH 4.5; Neutral: pH 5.9 and alkalic pH7.9.

For pre-treatment with citric acid, the citric acid (as 50% w/wsolution) was added to the oil at 70° C., kept stirred at 70° C. for 30minutes, subsequently the temperature was reduced to incubationtemperature and for the mildly acid condition the NaOH (as 2.0% w/wsolution) was added. In case of only NaOH addition, the oil was stirredat incubation temperature for 30 minutes.

During incubation, samples were taken after 0.5, 2, 4 and 24 hours, andanalysed by HPLC-Flu as described in Example 2, now against a set ofstandards with known concentration. Concentrations of all substrates(chlorophyll, pheophytins, pyropheophytin—a and b) and all reactionproducts (chlorophyllide, pheophorbide, pyropheophorbide—a and b) weresummed into total substrates and total reaction products, respectively,in mg/kg oil. All results are given in percentage of substrates andreaction products in the table below.

The results in Tables 3, 4 and 5 show that the Hordeum vulgare enzymeCHL26 according to SEQ ID NO: 1 has a wider application range in thepresence of acid and caustic and is active at a higher temperature thanthe reference chlorophyllase from Chlamydomonas reinhardtii.

TABLE 3 Chlorophyll derivatives (wt %) in crude canola oil afterincubation with the CHL26 enzyme from Hordeum vulgare or the referenceenzyme ELDC94 from Chlamydomonas reinhardtii at different conditions at40° C. CHL26 Reference Sum Sum 40° C. Time Sum reaction Sum reactionCondition [hr] substrates products substrates products — 0 94.9 5.1 94.95.1 Acidic 0.5 66.5 33.5 80.6 19.4 2 56.4 43.6 78.1 21.9 4 51.9 48.175.2 24.8 24 45.4 54.6 75.2 24.8 Mildly acidic 0.5 29.1 70.9 35.2 64.8 210.0 90.0 28.7 71.3 4 3.4 96.6 19.0 81.0 24 0.0 100.0 15.4 84.6 Neutral0.5 30.9 69.1 3.2 96.8 2 10.0 90.0 1.7 98.3 4 4.4 95.6 1.8 98.2 24 3.097.0 1.7 98.3 Mildly alkaline 0.5 71.8 28.2 60.7 39.3 2 72.5 27.5 55.244.8 4 57.4 42.6 38.2 61.8 24 14.8 85.2 32.8 67.2

TABLE 4 Chlorophyll derivatives (wt %) in crude canola oil afterincubation with the CHL26 enzyme from Hordeum vulgare or the referenceenzyme ELDC94 from Chlamydomonas reinhardtii at different conditions at50° C. CHL26 Reference Sum Sum 50° C. Time Sum reaction Sum reactionCondition [hr] substrates products substrates products — 0 94.9 5.1 94.95.1 Acidic 0.5 87.7 12.3 89.9 10.1 2 87.7 12.3 92.6 7.4 4 88.5 11.5 93.46.6 24 88.0 12.0 92.6 7.4 Mildly acidic 0.5 27.5 72.5 21.3 78.7 2 9.790.3 11.7 88.3 4 2.7 97.3 8.7 91.3 24 2.2 97.8 2.1 97.9 Neutral 0.5 28.571.5 5.8 94.2 2 13.7 86.3 3.9 96.1 4 5.5 94.5 2.0 98.0 24 0.6 99.4 0.699.4 Mildly alkaline 0.5 66.8 33.2 54.6 45.4 2 66.4 33.6 62.1 37.9 451.1 48.9 51.8 48.2 24 10.9 89.1 41.0 59.0

TABLE 5 Chlorophyll derivatives (wt %) in crude canola oil afterincubation with the CHL26 enzyme from Hordeum vulgare or the referenceenzyme ELDC94 from Chlamydomonas reinhardtii at different conditions at60° C. CHL26 Reference Sum Sum 60° C. Time Sum reaction Sum reactionCondition [hr] substrates products substrates products — 0 94.9 5.1 94.95.1 Acidic 0.5 90.5 9.5 94.7 5.3 2 90.5 9.5 95.0 5.0 4 90.7 9.3 92.7 7.324 90.5 9.5 94.9 5.1 Mildly acidic 0.5 13.5 86.5 60.4 39.6 2 2.5 97.565.7 34.3 4 4.1 95.9 66.4 33.6 24 2.0 98.0 68.5 31.5 Neutral 0.5 29.071.0 11.9 88.1 2 10.1 89.9 7.4 92.6 4 5.2 94.8 4.6 95.4 24 0.0 100.0 1.598.5 Mildly alkaline 0.5 57.7 42.3 52.5 47.5 2 65.1 34.9 80.4 19.6 467.4 32.6 80.4 19.6 24 33.0 67.0 93.0 7.0

Example 6 Incubation of Crude Canola Oil with an Enzyme fromChlamydomonas reinhardtii (ELDC94), Followed by Treatment with VariousSilicas

1,500 grams of crude canola oil was placed into a 2 liter jacketed glassbeaker with an overhead mixer with a square paddle and mixed at 90revolutions per minute (rpm). The jacket temperature was set at 65° C.20 mL of ELDC94 Chlamydomonas (alga) (prepared as described in Example4) and 100 grams of deionized water were added to the oil once the oiltemperature had reached the set point. The material was shear mixed for1 minute while covered with plastic wrap. The jacketed glass beaker wasmoved back to the overhead mixer and covered with plastic wrap. Thematerial was mixed covered for 24 hours at 250 rpm.

1.5 grams of 50% (wt. %) citric acid was added to the mixing oil. Theset point of the jacket was reduced to 55° C. Once the material reached55° C., the oil was moved to the shear mixer. 1.2 mL of 4 N NaOH wasadded to the oil and shear mixed 30 seconds. 0.3 grams of Purifine®Phospholipase C (PLC) and 30 grams of deionized water were added. Theoil was shear mixed for 1 minute while covered with plastic wrap. Thejacketed glass beaker was moved back to the overhead mixer and coveredagain with plastic wrap. The oil was mixed for 2 hours at 55° C. at 250rpm.

The beaker was moved back to the high shear mixer and 0.1 grams ofPhospholipase A1 (PLA1) enzyme (Lecitase Ultra) was added to the oil andshear mixed 1 minute while covered with plastic wrap. The jacketed glassbeaker was moved back to the overhead mixer and covered again withplastic wrap. The oil was mixed for 2 hours at 55° C. at 250 rpm.Increased the set point of the water bath to 75° C. Once the oil reached75° C., the oil was centrifuged utilizing Gyro-Centrifuge with the bowlwith holes closed. Samples of the oil were collected. The gums werediscarded.

The above reaction was repeated 11 times and the oil was combined andlabelled as “control”.

Six 500 gram samples of the above enzyme treated canola oil “control”were added to six 1000 mL round bottom flask. The oils were heated to80° C. and 0.25, 1.0, 2.0, 4.0, 6.0, and 8.0 grams of silica SP-2115were mixed into the oil and a vacuum of approximately 100 mbar wasadded. The temperature was increased to 100° C. and mixed for 30minutes. It was unexpected that the oil turned dark green during theadsorptive process with the test silica. In previous experiments usingindustrial silicas (TRISYL® (Grace Davison, Columbia, Md.), or, SORBSIL®silicas (INEOS Silicas, Joliet, Ill.) the color of the oil did notchange. The vacuum was broken and the material filtered with a BuchnerFunnel. The filter paper disc was a dark green color, but when usingindustrial silicas, the filter disc and cakes were always yellow. Thefilter disc and cake were a dark green color.

Two 500 gram samples of the above enzyme treated canola oil “control”from above were split and added to two 1000 mL round bottom flask withthe configuration of Adsorbent procedure. The oils were heated to 80° C.and 1.0 and 2.0 grams of silica SP-2116 were mixed into the oil and avacuum of approximately 100 mbar was added. The temperature wasincreased to 100° C. and mixed for 30 minutes. The color of the oilduring the trial did not change from the original color. The vacuum wasbroken and the material filtered with a Buchner Funnel. The filter discand cake were a yellow color.

Two 500 gram samples of the above enzyme treated canola oil “control”from above were split and added to two 1000 mL round bottom flask withthe configuration of Adsorbent procedure. The oils were heated to 80° C.and 1.0 and 2.0 grams of silica SP-2117 were mixed into the oil and avacuum of approximately 100 mbar was added. The temperature wasincreased to 100° C. and mixed for 30 minutes. The color of the oilduring the trial did not change from the original color. The vacuum wasbroken and the material filtered with a Buchner Funnel. The filter discand cake were a yellow color.

The content of the oils is set forth in Tables 6 and 7.

TABLE 6 P, Ca, Mg, and Fe content in ELDC94 treated oils followingfurther treatment with various silicas Phosphorus Calcium Magnesium Iron(ppm) (ppm) (ppm) (ppm) Crude Canola 836 161 119 1.6 Enzyme treated 14.211.42 1.99 0.42 Control (ELDC94) SP-2115-0.25 g 9.34 6.19 1.19 0.19SP-2115-1 g 9.34 7.32 1.43 0.25 SP-2115-2 g 4.52 3.86 1.17 0.13SP-2115-4 g 3.24 3.26 0.90 0.14 SP-2115-6 g 5.24 4.37 1.53 0.16SP-2115-8 g 1.96 2.12 0.88 0.05 SP-2116-0.25 g 9.99 6.81 1.25 0.21SP-2116-1 g 9.43 7.01 1.42 0.22 SP-2117-0.25 g 10.06 7.32 1.31 0.23SP-2117-1 g 9.55 7.81 1.57 0.16

TABLE 7 Content of chlorophyll derivatives in ELDC94 treated oilsfollowing further treatment with various silicas Chlorophyll andChlorophyll Derivatives “A” “B” CHYL PYN PPYN POB PPOB CHYL PYN TotalUV/Vis (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) CrudeCanola 0.31 5.48 11.72 0.06 0.94 0.56 0.79 19.86 53.60 Control b.d. 0.492.79 1.49 2.95 b.d. 0.34 8.06 38.49 SP-2115 - 0.25 g b.d. 0.47 2.73 1.3 2.95 b.d. 0.46 7.91 36.29 SP-2115 - 1 g b.d. 0.31 1.65 0.98 1.62 b.d.b.d. 4.56 25.65 SP-2115 - 2 g b.d. 0.2  1.43 b.d. 0.89 b.d. b.d. 2.5220.36 SP-2115 - 4 g b.d. b.d. 0.83 b.d. 0.61 b.d. b.d. 1.44 14.32SP-2115 - 6 g b.d. 0.39 0.68 0.32 b.d. b.d. b.d. 1.39 11.04 SP-2115 - 8g b.d. 0.34 0.58 b.d. b.d. b.d. b.d. 0.92 8.93 SP-2116 - 0.25 g b.d.0.41 2.78 1.59 3.39 b.d. 0.44 8.61 39.03 SP-2116 - 1 g b.d. 0.52 2.461.42 2.99 b.d. 0.39 7.78 36.51 SP-2117 - 0.25 g b.d. 0.38 2.53 1.4  3.04b.d. 0.34 7.69 38.66 SP-2117 - 1 g b.d. 0.53 2.48 1.43 3.01 b.d. 0.397.84 36.66 Control deodorized b.d. 0.11 1.02 1.58 n.d. b.d. b.d. 2.7119.31 SP-2115 - 6 g b.d. b.d. b.d. b.d. b.d. b.d. b.d. b.d. 3.84deodorized CHYL = Chlorophyll; PYN = Pheophytin; PPYN = Pyropheophytin;POB = Pheophorbide; PPOB = Pyropheophorbide b.d. = below detection n.d.= not determined

ELDC94 Chlamydomonas (alga) enzyme decreases the amount of chlorophylland chlorophyll derivates from 19.86 ppm to 8.06 ppm (53.6 to 38.49 ppmvia the AOCS UV/VIS method) after 24 hours. However, it is not greatenough to significantly enable the process in an industrial process. Anenzyme with a greater ability to hydrolyze chlorophyll and chlorophyllderivatives is required as well as a process for greater removal ofthose generated derivatives.

The above data demonstrates that silica SP-2115 has the greatestcapacity to remove metals, chlorophyll, and chlorophyll derivativescompared to the other two silicas. The UV/Vis method demonstrates thattreatment with the low doses (i.e., 0.25 g and 1 g) of SP-2115 reducesthe chlorophyll by 2.20 and 12.84 ppm, respectively, as compared to theamount of chlorophyll in the control (i.e., 38.49 ppm). In comparison,the amount of chlorophyll following treatment with the lowest dosages(i.e., 0.25 g) of SP-2116 and SP-2117 increased by 0.54 ppm and 0.17ppm, respectively, while the amount of chlorophyll following treatmentwith the highest dosages (i.e., 1 g) of SP-2116 and SP 2117 decreased1.98 ppm and 1.83 ppm, respectively, as compared to control. The HPLCtest method demonstrates the same pattern of limited reduction at thehighest dosage for SP-2116 and SP-2117. The data also demonstrates thatthe HPLC method for chlorophyll and chlorophyll derivatives needs to beimproved by finding additional standards and response factors in orderto bring it closer in line with the AOCS method for measuring the greencolor in vegetable oils. Additional work on the method has beencompleted and is encompassed in the following examples.

Example 7 Incubation of Solvent Extracted Crude Canola Oil with anEnzyme Having Pyropheophytinase Activity Derived from Hordeum vulgare(CHL26) Compared to a Reference Enzyme from Chlamydomonas reinhardtii(ELDC94)

A 35-pound container of solvent extracted crude canola oil was pouredinto large stainless-steel container and made uniform with IKA mixer.

After mixing, approximately 1.5 kg of crude canola was placed into a 2liter jacketed glass beaker with an overhead mixer with a square paddleand mixed at 90 revolutions per minute (rpm). The jacket temperature wasset at 65° C. 0.7 grams of enzyme ELDC94 (reaction 1) or 7.5 grams ofCHL26 (reaction 2), produced as described in Example 4, were added tothe oil together with 100 grams of deionized water once the oiltemperature had reached the set point. The material was shear mixed for1 minute while covered with plastic wrap. The jacketed glass beaker wasmoved back to the overhead mixer and covered with plastic wrap. Thematerials were incubated with the enzymes for 24 hours at 250 rpm.

1.5 grams of 50% (wt. %) citric acid was added to the mixing oil. Theset point of the jacket was reduced to 55° C. Once the material reached55° C., the oil was moved to the shear mixer. 1.2 mL of 4 N NaOH wasadded to the oil and shear mixed 30 seconds. 0.3 grams of Purifine®Phospholipase C (PLC) and 30 grams of deionized water were added. Theoil was shear mixed for 1 minute while covered with plastic wrap. Thejacketed glass beaker was moved back to the overhead mixer and coveredagain with plastic wrap. The oil was mixed for 2 hours at 55° C. at 250rpm.

The beaker was moved back to the high shear mixer and 0.1 grams of afungal phospholipase A₁ (PLA₁) enzyme was added to the oil and shearmixed 1 minute while covered with plastic wrap. The jacketed glassbeaker was moved back to the overhead mixer and covered again withplastic wrap. The oil was mixed for 2 hours at 55° C. at 250 rpm.Increased the set point of the water bath to 75° C. Once the oil reached75° C., the oil was centrifuged utilizing a Gyro-Centrifuge with thebowl with holes closed. Samples of the oil and gums were collected andanalysed for the presence of P, Ca, Mg and Fe and chlorophyllderivatives (using HPLC) as described above.

The mixture of oil and heavy phase remaining in the centrifuge bowl werepoured in to a 400 mL beaker where the oil was decanted off. Theremaining oil and heavy phase were placed into 50 mL centrifuge tubesand spun. The oil from the decanted bowl and in the tubes was discardedand liquid heavy phases were combined.

The results in Table 8 and FIG. 4 a) show that the CHL26 enzyme havingpyropheophytinase activity according SEQ ID NO: 1 is able to reducechlorophyll derivatives in solvent extracted crude canola oil.Chlorophyll substrates are chlorophyll, pheophytin, and pyropheophytinand chlorophyll products are chlorophyllide, pheophorbide andpyropheophorbide.

TABLE 8 Compounds (in ppm) in crude canola oil after treatment withenzymes CHL26 and the reference enzyme ELDC94 Chlorophyll derivatives(HPLC) (ppm) Enzyme P Ca Mg Fe Total Substrates Products None* 903.0243.0 127 9.89 15.40 14.72 0.50 ELDC94 88.5 80.9 14.6 1.49 8.39 0.218.18 CHL26 82.0 77.3 14.1 1.58 9.15 1.26 7.89 *Starting material (crudecanola oil)

The results in FIG. 4 b) show that there are still unreactedphospholipids present in the collected heavy phase, which is anindication that the phospholipase reactions were too short to come tocompletion.

Example 8 Incubation of Pressed Crude Canola Oil with the CHL26 Enzymeat Varying Conditions, and Treatment with Silica SP-2115

A 35-pound container of pressed crude canola oil was poured into largestainless-steel container and made uniform with IKA mixer

Reaction 3—CHL26 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr,Followed by a 2 hr Incubation with PLA1

About 1.5 kg of crude canola was placed into a 2 liter jacket glassbeaker with an overhead mixer with a square paddle. The oil was mixed at90 rpm. The jacket temperature was set at 70° C. 1.5 grams of 50% (wt.%) citric acid was added to the mixing oil and shear mixed 1 minute. Theset point of the jacket was reduced to 60° C. Once the material reached60° C., the oil was moved to the shear mixer. 1.2 mL of 4 N NaOH wasadded to the oil and shear mixed 30 seconds. 0.3 grams of Purifine PLC(LR79.14 February 2018), 0.02 grams of Purifine PI-PLC, 7.5 grams ofCHL26 enzyme [Hordeum vulgare var. distichum (barley, plant)], producedas described in Example 4, and 100 grams of deionized water. Thematerial was shear mixed for 1 minute while covered with plastic wrap.The jacketed glass beaker was moved back to the overhead mixer andcovered again with plastic wrap. The oil was mixed for 2 hours at 60° C.at 250 rpm.

The jacketed glass beaker was again moved to the hear mixer where 0.075grams of PLA, (notebook, 074362) was added and the oil was shear mixed 1minute. The jacketed glass beaker was moved back to the overhead andcovered with plastic wrap. The oil was mixed and the reactions wereallowed to continue for 2 hours at 250 rpm. Increased the set point ofthe water bath to 75° C. Once the oil reached 75° C., the oil wascentrifuged utilizing Gyro-Centrifuge with the bowl with holes closed.Samples of the oil and gums were collected.

The mixture of oil and heavy phase remaining in the centrifuge bowl werepoured in to a 400 mL beaker where the oil was decanted off. Theremaining oil and heavy phase were placed into 50 mL centrifuge tubesand spun. The oil from the decanted bowl and in the tubes was discardedand liquid heavy phases were combined.

Reaction 4—ELDC94 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr,Followed by a 2 hr Incubation with PLA₁

The same procedure from reaction 1 above was employed for enzyme ELDC94,using 0.61 grams of the formulated enzyme solution (produced asdescribed in Example 4).

Two 450 gram samples of the Reaction 4 enzyme treated canola oil weresplit and added to two 1000 mL round bottom flask with the configurationof Adsorbent procedure. The oils were heated to 80° C. and 1.0 and 2.0grams of silica SP-2115 were mixed into the oil and a vacuum ofapproximately 100 mbar was added. The temperature was increased to 100°C. and mixed for 30 minutes. The oil turned dark green during theadsorptive process with the test silica. The vacuum was broken and thematerial filter with a Buchner Funnel. The filter disc and cake were adark green color.

Reaction 5—CHL26 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr,Followed by a 4 hr Incubation with PLA₁

The same procedure was followed as reaction 1, but the PLA1 reaction wasallowed to react for 4 hours instead of only 2 hours.

Two 450 grams samples of the Reaction 5 enzyme treated canola oil weresplit and added to two 1000 mL round bottom flask with the configurationof Adsorbent procedure. The oils were heated to 80° C. and 2.0 and 3.0grams of silica SP-2115 were mixed into the oil and a vacuum ofapproximately 100 mbar was added. The temperature was increased to 100°C. and mixed for 30 minutes. The vacuum was broken and the materialfilter with a Buchner Funnel. The filter disc and cake were a dark greencolor

Reaction 6—CHL26 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr,Followed by a 4 hr Incubation with PLA₁

The same procedure was followed as reaction 3, except twice the amountof CHL26 (15 grams total) was added to the reaction.

Two 450 grams samples of the Reaction 6 enzyme treated canola oil weresplit and added to two 1000 mL round bottom flask with the configurationof Adsorbent procedure. The oils were heated to 80° C. and 1.0 and 2.0 gof silica SP-2115 were mixed into the oil and a vacuum of approximately100 mbar was added. The temperature was increased to 100° C. and mixedfor 30 minutes. The vacuum was broken and the material filter with aBuchner Funnel. The filter disc and cake were a dark green color.

Reaction 7—CHL26 Incubation with PLC and PI-PLC at Neutral pH for 2 hr,Followed by a 4 hr Incubation with PLA₁

The same procedure was followed as reaction 1, except no pH adjustmentwas made.

Reaction 8—SBO CHL26 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr,Followed by a 2 hr Incubation with PLA₁

The same procedure as reaction 1 was followed, but the oil was a solventextracted crude soybean oil (SBO).

Three 450 g samples of the Reaction 8 enzyme treated soybean oil weresplit and added to three 1000 mL round bottom flask with theconfiguration of Adsorbent procedure. The oils were heated to 80° C. and0.25, 0.5 and 1.0 grams of silica SP-2115 were mixed into the oil and avacuum of approximately 100 mbar was added. The temperature wasincreased to 100° C. and mixed for 30 minutes. The vacuum was broken andthe material filter with a Buchner Funnel. The filter disc and cake werea dark green color.

In Table 9 and FIG. 5 b ), the phosphorus (P), calcium (Ca), magnesium(Mg), and iron (Fe) contents of the oils and the respective gums beforeand after enzyme treatments according to reactions 1 to 6 are shown. Atneutral pH, a higher amount of P remained in the oil as compared toreaction at pH 4.5. Table 9 also shows P, Ca, Mg, and Fe contents of theoils following treatment with silica SP-2115. This data demonstrates thesilica treatment removes trace phosphorus and metals to levelssufficient to meet industrial standards for bleached oils without theuse of bleaching earth. They did not lose their capacity to adsorb theseimpurities when the MgO was added.

The results in Table 10 and FIG. 5 a ) show that the CHL26 enzymeconverts a higher amount of chlorophyll derivatives in crude canola oilas compared to ELDC94, when the enzymes are incubated under the sameconditions (reactions 1 and 2). Table 10 also shows that contacting theenzyme treated oils with silica SP-2115 reduces the amount of bothchlorophyll substrates and chlorophyll products in the oils, as comparedthe amount of chlorophyll substrates and products in enzyme treated oilsthat are not further contacted with the silica.

In the present example the CHL26 enzyme converted a higher amount ofchlorophyll substrates into the respective chlorophyll products in crudecanola oil under neutral conditions as compared to acid conditions (pH4.5) (compare reaction 7 with reactions 3, 5 and 6).

The CHL26 enzymes also converts chlorophyll substrates in soybean oilinto the respective chlorophyll products (reaction 8).

The results in Table 10 also show that a higher amount of chlorophyllproducts were found in the gums (heavy phase) when the oil was reactedwith the CHL26 enzyme as compared to the reaction with the ELDC94enzyme.

TABLE 9 Compounds in canola oil (Can) or soybean oil (SBO) aftertreatment with the CHL26 enzyme compared to reference enzyme ELDC94and/or no enzyme treatment and/or after silica treatment Silica SP- 2115P Ca Mg Fe Reaction Oil pH (grams) (ppm) None, Crude Can — — 210 90.536.7 0.90 Rxn 3 - CHL26 Can 4.5 — 10.7 7.9 1.7 0.20 Rxn 4 - ELDC94 Can4.5 — 4.4 3.3 0.9 0.07 Rxn 4 1 1.5 2.3 0.3 0.05 Rxn 4 2 1.4 2.3 0.3 0.05None, Crude Can — — 210 90.5 36.7 0.90 Rxn 5 - CHL26 Can 4.5 — 3.9 2.80.6 0.16 Rxn 5 2 1.6 1.8 0.5 0.10 Rxn 5 3 b.d. 0.3 Tr 0.02 Rxn 6 - CHL26Can 4.5 — 2.0 1.5 0.4 0.10 Rxn 6 1 0.5 0.9 0.2 0.07 Rxn 6 2 0.6 0.8 0.20.06 Rxn 7 - CHL26 Can Neu- — 103 80.9 10.5 0.88 tral None, Crude SBO —— 773 66.2 64.3 0.76 Rxn 8 - CHL26 SBO 4.5 — 5.8 0.5 0.7 0.04 Rxn 8  0.25 0.6 0.2 0.1 0.03 Rxn 8   0.5 0.7 0.2 0.2 0.03 Rxn 8 1 b.d. 0.10.1 0.02 b.d.—below detection Tr—trace

TABLE 10 Chlorophyll derivatives in canola oil or soybean oil and theseparated gums after treatment with the CHL26 enzyme compared toreference enzyme ELDC94 and/or no enzyme treatment and/or after silicatreatment Chlorophyll Chlorophyll derivatives derivatives in the oil inthe gums (ppm) (ppm) Oil Substrates Products Substrates Products CrudeCanola 13.13 0.90 — — Rxn 3-CHL26, pH 4.5 4.19 7.97 0.06 6.39 Rxn4-ELDC94, pH 4.5 10.28 2.62 0.18 3.06 Rxn 4-SP-2115, 1 g 3.46 b.d. — —Rxn 4-SP-2115, 2 g 6.27 0.22 — — Crude Canola 13.13 0.90 — — Rxn 5,CHL26, pH 4.5 6.85 5.69 0.30 2.68 Rxn 5-SP-2115, 2 g 4.38 0.57 — — Rxn5-SP-2115, 3 g 0.78 b.d. — — Rxn 6-CHL 26, pH 4.5 6.01 6.41 0.19 1.99Rxn 6-SP-2115, 1 g 3.88 1.22 — — Rxn 6-SP-2115, 2 g 2.77 0.53 — — Rxn7-CHL26, neutral pH 1.26 10.02 b.d. 5.02 Crude SBO 0.31 b.d. — — Rxn8-CHL26, pH 4.5 0.28 b.d. b.d. 0.56 Rxn 8-SP-2115, 0.25 g 0.24 b.d. — —Rxn 8-SP-2115, 0.5 g 0.14 b.d. — — Rxn 8-SP-2115, 1 g 0.87 b.d. — —b.d.—below detection,

Example 9 Use of CHL26 Enzyme and Silica Treatment in Caustic RefiningApplication of Canola Oil and Soybean Oil

The following experiments are an evaluation of the CHL26 in a causticrefining application where the oil has been treated with a phosphoricacid and sodium hydroxide, as occurs in industrial processes of canolaand soybean oils. A “once refined” product is an oil that was treatedwith phosphoric acid, then treated with sodium hydroxide to convert theFree Fatty Acids (FFA) into sodium soaps that are water soluble andremoved in water or “heavy” phase of the “refining” centrifuge. The oilwas then washed with water (2 to 10 percent w/w) to remove the remainingsoaps and residual phospholipids present in the oil. Optionally, theenzymes were evaluated after the refining centrifuge in the waterwashing step, but at a much lower temperature.

A five-gallon plastic pail of Once Refined Canola (ORCAN) oil was mixedwith a high shear mixer to make uniform. 2-3 kg samples were pulled foruse in the experiments below.

Reaction 9—ELDC94-Comparative

2 kg of once refined canola was placed into a 4 liter glass beaker on ahot plate with overhead mixing at 90 rpm. The oil was heated to 60° C.under agitation. Once the material reached 60° C., the beaker was movedto the shear mixer. 0.8 grams of enzyme ELDC94 (produced as described inExample 4) and 100 grams of deionized water were added to the oil. Thematerial was shear mixed for 1 minute while covered with plastic wrap tominimize water loss. The glass beaker was moved back to the overheadmixer and covered with plastic wrap. The oil was mixed for 4 hours at60° C. at 250 rpm. The temperature was increased to 75° C. The oil wascentrifuged utilizing Gyro-Centrifuge. The separated oil was collected.

The mixture of oil and heavy phase remaining in the centrifuge bowl werepoured in to a 400 mL beaker where the oil was decanted off. Theremaining oil and heavy phase were placed into 50 mL centrifuge tubesand spun. The oil from the decanted bowl and in the tubes was discardedand liquid heavy phases were combined. The heavy phase was a dark green.

Reaction 10—ELDC94-Comparative

Reaction 10 was a repeat of reaction 9, except 3 kg of oil was used and2.0 grams of ELDC94 (produced as described in Example 4).

After the analyses of the oils from reaction 9 and 10, the oils werecombined mixed and analysed again.

Reaction 11—CHL26

Reaction 11 was a repeat of reaction 9, except that 10.1 grams of CHL26(produced as described in Example 4) was used instead of ELDC94. Theheavy phase was a lighter green than reactions 9 and 10.

Reaction 12—CHL26

Reaction 12 was a repeat of reaction 10, except that 20 grams of CHL26was utilized.

After analyses, the oils of reaction 11 and 12 were combined and mixedand after mixing analysed again.

Reaction 13—ELDC94-Comparative

3 kg of once refined soybean oil (ORSBO) was pulled from a causticrefining production line number 1 after the water washing centrifuge.The oil was placed into a 4 liter glass beaker and placed onto a hotplate with overhead mixing with a square mixing paddle (90 rpm). Oncethe material cooled 60° C., the beaker was moved to a shear mixer. 1.0grams of ELDC94 enzyme produced as described in Example 4) and 150 gramsof deionized water were added to the oil. The material was shear mixedfor 1 minute while covered with a plastic wrap to minimize moistureloss. The glass beaker was moved back to the overhead mixer and againcovered with a plastic wrap. The oil was mixed for 4 hours at 60° C. at250 rpm. The temperature was increased to 75° C. and then the oil wascentrifuged utilizing Gyro-Centrifuge.

Collected oil and heavy samples for further analyses.

The remaining oil and heavy phase remaining in the centrifuge bowl werepoured in to a 400 mL beaker where the oil was decanted off. Theremaining oil and heavy phase were placed into 50 mL centrifuge tubesand spun. The remaining oil in the tubes was discarded and liquid heavyphases were combined. The heavy phase was colorless, no discerniblecolor pigments.

Reaction 14—CHL26

Reaction 14 was a repeat of reaction 13, except that 15 grams of CHL26(produced as described in Example 4) was utilized instead of ELDC94.

Reaction 15—EDLC94-Comparative

3 kg grams of once refined soybean oil (ORSBO) was pulled from a causticrefining production line number 1 after the water washing centrifuge.The oil was placed into a 4 liter glass beaker and placed onto a hotplate with overhead mixing with a square mixing paddle (90 rpm). Oncethe material cooled 60° C., the beaker was moved to a shear mixer. 1.2grams of ELDC94 enzyme produced as described in Example 4 and 150 gramsof deionized water were added to the oil. The material was shear mixedfor 1 minute while covered with a plastic wrap to minimize moistureloss. The glass beaker was moved back to the overhead mixer and againcovered with a plastic wrap. The oil was mixed for 4 hours at 60° C. at250 rpm. The temperature was increased to 75° C. and then the oil wascentrifuged utilizing Gyro-Centrifuge.

Collected oil and heavy phase (gums) samples for further analyses.

The remaining oil and heavy phase remaining in the centrifuge bowl werepoured in to a 400 mL beaker where the oil was decanted off. Theremaining oil and heavy phase were placed into 50 mL centrifuge tubesand spun. The remaining oil in the tubes was discarded and liquid heavyphases were combined. The heavy phase was colorless, no discerniblecolor pigments.

Reaction 16—CHL26

Reaction 16 was a repeat of reaction 15, except that 15 grams of CHL26was utilized.

The results of reactions 9 to 16 and the results of the combined andmixed oils from reaction 9 and 10 and from reactions 11 and 12 are shownin Table 11 and FIGS. 6-6A.

The results in Table 9 and FIGS. 6-6A show that the enzyme CHL26, havingpyropheophytinase converts a higher amount of chlorophyll substrates(chlorophyll, pheophytin and pyropheophytin) to its chlorophyll products(chlorophyllide, pheophorbide, pyropheophorbide) than the referencechlorophyllase enzyme ELDC94.

TABLE 11 Chlorophyll derivatives (substrates and products) in oncerefined canola oil (ORCAN) and once refined soybean oil (ORSBO) aftercaustic refining and after treatment with the CHL26 enzyme and theELDC94 (reference) enzyme Chlorophyll derivatives in oil (ppm) Enzymereaction Substrates Products None: ORCAN 27.38 b.d. Rxn 9-ELDC94 7.874.99 Rxn 10-ELDC94 19.37 0.42 Combined 9 & 10 18.71 0.39 Rxn 11-CHL2611.60 4.96 Rxn 12-CHL26 n.m. n.m. Combined 11 & 12 12.00 6.19 None:ORSBO 3.85 b.d. Rxn 13-ELDC94 1.09 0.06 None: ORSBO 3.90 b.d. Rxn14-CHL26 1.12 0.18 None: ORSBO 3.90 b.d. Rxn 15-ELDC94 2.05 b.d. Rxn16-CHL26 1.77 0.07 b.d. = below detection n.m. = not measured

The results of Table 12 show the contents of free fatty acids (FFA),soap and phosphor and Ca, Mg, Fe, and/or chlorophyll in once refinedcanola oil and once refined soybean oil after enzymatic treatmentsdescribed above.

TABLE 12 Composition of once refined canola oil (ORCAN), once refinedsoybean (ORSBO) oil after caustic refining and after treatment with theCHL26 enzyme and the ELDC94 (reference) enzyme Lovibond FFA Soap P Ca MgFe UV/Vis HPLC * Color (%) (ppm) (ppm) (ppm) (ppm) (ppm) (ppb) (ppm) RedYellow ORCAN 0.05 195 4.5 0.9 0.2 0.03 12047 27.38 t.d. t.d. Rxn 9 -0.05 b.d. 0.5 0.7 tr 0.02 10461 12.86 t.d. t.d. ELDC94 Rxn 10 - 0.07b.d. 0.6 1.8 tr 0.03  9377 19.70 t.d. t.d. ELDC94 Combined 0.06 b.d. 0.60.7 tr 0.07 — — — — 9 & 10 Rxn 11 - 0.06 tr 1.6 2.4 0.1 0.11 11079 16.56t.d. t.d. CHL26 Rxn 12 - 0.06 b.d. 1.7 2.9 0.1 0.07 11536 n.m. t.d. t.d.CHL26 Combined 0.06 tr 1.7 2.7 0.1 0.09 — — — — 11 & 12 ORSBO 0.12  200.3 0.2 b.d. b.d. — — — — Rxn 13 - 0.10 b.d. 0.2 0.1 b.d. b.d. — — — —ELDC94 ORSBO 0.06  27 1.0 0.4 tr b.d. — — — — Rxn 14 - 0.05 b.d. 0.2 0.1b.d. b.d. — — — — CHL26 ORSBO 0.03 242 2.8 0.7 0.2 tr — — — — Rxn 15 -0.02 tr 0.3 0.2 b.d. 0.1  — — — — ELDC94 ORSBO 0.05 396 3.3 0.9 0.2 b.d.— — — — Rxn 16 - 0.03 tr b.d. 0.2 b.d. b.d. — — — — CHL26 tr = traceb.d. = below detection n.m. = not measured t.d. = too dark to measure *= HPLC was a measurement of total chlorophyll derivatives

Reactions 9 and 10—Silica Treatment

Oils from reactions 9 and 10 were combined and then split into three 500gram samples of the enzyme treated ORCAN oil and added to three 1000 mLround bottom flask with the configuration of Adsorbent procedure. Theoils were heated to 80° C. and 1.0, 2.0 and 3.0 grams of silica SP-2115were mixed into the oil and a vacuum of approximately 100 mbar wasadded. The temperature was increased to 100° C. and mixed for 30minutes. The oil turned dark green during the adsorptive process withthe test silica. The vacuum was broken and the material filter with aBuchner Funnel. The filter paper disc was a dark green color. The oilswere labeled as 9101, 9102, and 9103 respectively. Oil labeled as 9100was the sample of reaction 9 and reaction 10 combined.

The canola oil treated, 9102 (436 grams) and 9103 (448 grams) werecombined and placed in a 3 L Claisen flask. The oil was sparged withnitrogen for approximately 2 minutes. The vacuum was initiated and thenitrogen sparge was discontinued and water vapor from the steamgenerator was allowed to begin the deodorization process. The vacuumachieved was between 0.82-0.98 mBar during the deodorization process.The oil was heated under vacuum and water sparge (3 wt. %) to 230° C.The sparge and temperature were maintained for two hours. The heat wasdiscontinued and the oil was allowed to cool under vacuum and watersparge. The vacuum was broken with nitrogen at approximately 100° C. andallowed to further cool to 70° C. before opening to the air. The oil wasa dark greenish/grey tint. The oil was labeled as 91023-DEO.

The results in Table 13 show the contents of free fatty acids (FFA),soap, P, Ca, Mg, Fe, and chlorophyll in the combined reaction 9 and 10oils following silica treatment as described above.

TABLE 13 Composition of once refined canola oil (ORCAN), after enzymetreatment or after enzyme and silica treatment Lovibond FFA Soap P Ca MgFe UV/Vis HPLC* Color (%) (ppm) (ppm) (ppm) (ppm) (ppm) (ppb) (ppm) RedYellow 9100 0.06 b.d. 0.6 0.7 tr 0.07 n.m. 19.10 t.d. t.d. 9101 n.m.b.d. 0.4 0.3 b.d. tr 8273 19.52 8.0 70 9102 n.m. n.m. 0.3 0.2 b.d. b.d.6848 17.13 7.6 70 9103 n.m. n.m. b.d. 0.1 b.d. b.d. 5514 14.04 7.1 7091023-Deo 0.02 n.m. n.m. n.m. n.m. n.m. 1833 3.20 n.m. n.m. tr = traceb.d. = below detection n.m. = not measured t.d. = too dark to measure *=HPLC was a measurement of total chlorophyll derivatives

The results in Table 14 show the chlorophyll substrates and products inthe combined reaction 9 and 10 oils following silica treatment asdescribed above.

TABLE 14 Chlorophyll substrate and product composition in oils afterenzyme and silica treatment a a′ b CHYL PYN PPYN POB PPOB PYN POB CHYLPYN ppm ppm ppm ppm ppm ppm ppm ppm ppm 9100 0.19 5.97 6.85 0.18 0.151.85 0.05 0.12 1.64 9101 0.10 4.78 5.85 0.03 0.02 3.31 b.d. 0.11 1.329102 0.10 4.50 4.14 b.d. b.d. 3.07 b.d. 0.11 1.34 9103 0.10 3.73 2.73b.d. b.d. 2.65 b.d. 0.11 1.23 91023-Deo 0.05 0.48 1.43 b.d. b.d. b.d.b.d. 0.17 0.12 b b′ Decolorase PPYN POB PPOB CHYL PYN POB Total Sub.Prod. ppm ppm ppm ppm ppm ppm ppm ppm ppm 9100 1.58 b.d. b.d. b.d. 0.50b.d. 19.10 18.71 0.39 9101 3.14 b.d. b.d. b.d. 0.85 b.d. 19.52 19.460.06 9102 2.98 b.d. b.d. b.d. 0.90 b.d. 17.13 17.13 b.d. 9103 2.65 b.d.b.d. b.d. 0.86 b.d. 14.04 14.04 b.d. 91023-Deo 0.96 b.d. b.d. b.d. b.d.b.d. 3.20 3.20 b.d. CHYL = Chlorophyll; PYN = Pheophytin; PPYN =Pyropheophytin; POB = Pheophorbide; PPOB = Pyropheophorbide; Sub =Substrates; Prod = Products b.d. = below detection

The feed material from the combined samples from reactions using ELDC94of 18.71 ppm as reported from the HPLC method shows a dramatic reductionin substrates following treatment with the SP-2115 to 14.04 ppm and acomplete removal of the products from 0.39 ppm in the combined samplesto below detection limit following treatment with SP-2115.

Reactions 11 and 12—Silica Treatment

Oils from reactions 11 and 12 were combined and then split into three500 gram samples of the enzyme treated ORCAN oil and added to three 1000mL round bottom flask with the configuration of Adsorbent procedure. Theoils were heated to 80° C. and 1.0, 2.0 and 3.0 grams of silica SP-2115were mixed into the oil and a vacuum of approximately 100 mbar wasadded. The temperature was increased to 100° C. and mixed for 30minutes. The vacuum was broken and the material filter with a BuchnerFunnel. The filter disc and cake were a dark green. The oils werelabeled as 11121, 11122, and 11123 respectively. Oil labeled as 11120was the combined sample of reaction 11 and reaction 12 oil.

The canola oil treated, 11122 (449 grams) and 11123 (451 grams) werecombined and placed in a 3 L Claisen flask and assembled according thedeodorization procedure. The oil was sparged with nitrogen forapproximately 2 minutes. The vacuum was initiated and the nitrogensparge was discontinued and water vapor from the steam generator wasallowed to begin the deodorization process. The vacuum achieved wasbetween 0.28-0.56 mBar during the deodorization process. The oil washeated under vacuum and water sparge (3 wt. %) to 230° C. The sparge andtemperature were maintained for two hours. The heat was discontinued andthe oil was allowed to cool under vacuum and water sparge. The vacuumwas broken with nitrogen at approximately 100° C. and allowed to furthercool to 70° C. before opening to the air. The oil was a lightgreenish/grey tint. The oil was labeled as 111223-DEO.

The results in Table 15 show the contents of free fatty acids (FFA),soap, P, Ca, Mg, Fe, and chlorophyll in the combined reaction 11 and 12oils following silica treatment as described above.

TABLE 15 Composition of once refined canola oil (ORCAN), after enzymetreatment or after enzyme and silica treatment Lovibond FFA Soap P Ca MgFe UV/Vis HPLC* Color (%) (ppm) (ppm) (ppm) (ppm) (ppm) (ppb) (ppm) RedYellow 11120 0.06 tr 1.7 2.7 0.1 0.09 n.m. 18.19 t.d. t.d. 11121 n.m.b.d. 1.2 1.8 tr 0.07 6180 12.05 8.1 70 11122 n.m. n.m. 0.8 1.1 b.d. 0.034267 9.38 8.9 70 11123 n.m. n.m. 0.2 0.3 b.d. 0.01 3576 4.12 7.1 70111223-DEO 0.02 n.m. n.m. n.m. n.m. n.m. 1741 2.41 n.m. n.m. tr = traceb.d. = below detection n.m. = not measured t.d. = too dark to measure *=HPLC was a measurement of total chlorophyll derivatives

The results in Table 16 show the chlorophyll substrates and products inthe combined reaction 11 and 12 oils following silica treatment asdescribed above.

TABLE 16 Chlorophyll substrate and product composition in oils afterenzyme treatment or after enzyme and silica treatment a a′ b CHYL PYNPPYN POB PPOB PYN POB CHYL PYN ppm ppm ppm ppm ppm ppm ppm ppm ppm 111200.10 2.22 3.18 3.03 1.29 3.16 0.28 0.12 0.73 11121 0.10 2.40 2.98 0.430.21 2.49 0.08 0.12 0.67 11122 0.10 2.26 1.97 0.12 0.05 1.96 0.04 b.d.0.63 11123 0.09 0.98 0.68 0.05 0.04 0.85 b.d. b.d. 0.31 111223-DEO 0.040.40 1.12 b.d. b.d. b.d. b.d. 0.16 b.d. b b′ Decolorase PPYN POB PPOBCHYL PYN POB Total Sub. Prod. ppm ppm ppm ppm ppm ppm ppm ppm ppm 111201.73 0.86 0.64 0.12 0.64 0.08 18.19 12.00 6.19 11121 1.72 0.13 0.10 b.d.0.60 0.03 12.05 11.07 0.98 11122 1.57 0.04 0.06 b.d. 0.58 b.d. 9.38 9.060.31 11123 0.74 0.03 0.05 b.d. 0.29 b.d. 4.12 3.95 0.17 111223-DEO 0.69b.d. b.d. b.d. b.d. b.d. 2.41 2.41 b.d. CHYL = Chlorophyll; PYN =Pheophytin; PPYN = Pyropheophytin; POB = Pheophorbide; PPOB =Pyropheophorbide; Sub = Substrates; Prod = Products b.d. = belowdetection

The feed material from the combined samples from reactions using CHL26of 18.19 ppm as reported from the HPLC method shows a dramatic reductionin substrates following treatment with the SP-2115 of from 12.00 to 3.95ppm, and a reduction of the products from 6.19 ppm to 0.17 ppm. It isclear that SP-2115 has a capacity for both the substrates and productsof the enzymatic reaction of CHL26 for their removal in an adsorptiveprocess.

Reactions 5 and 13—Silica Treatment. Comparison of SP-2114 and SP-2115

Three 500 gram samples of the enzyme treated refined soybean oil fromreaction 13 were split and added to three 1000 mL round bottom flaskwith the configuration of Adsorbent procedure. The oils were heated to80° C. and 0.5, 1.0 and 2.0 grams of silica SP-2115 were mixed into theoil and a vacuum of approximately 100 mbar was added. The temperaturewas increased to 100° C. and mixed for 30 minutes. The vacuum was brokenand the material filter with a Buchner Funnel. The filter disc and cakewere a dark green color. The oils were labeled as 135, 1310, and 1320respectively. Oil labeled as 130 was the sample from reaction 13.

A 500 gram sample of the enzyme treated refined soybean oil fromreaction 5 was added to a 1000 mL round bottom flask with theconfiguration of Adsorbent procedure. The oil was heated to 80° C. and2.0 grams of silica SP-2114 was mixed into the oil and a vacuum ofapproximately 100 mbar was added. The temperature was increased to 100°C. and mixed for 30 minutes. The color change was greenish/brown in theoil was observed during the adsorption process. The vacuum was brokenand the material filter with a Buchner Funnel. The filter disc and cakewere light green. The sample was labeled as 132-2114.

The results in Table 17 show the contents of free fatty acids (FFA),soap, P, Ca, Mg, Fe, and chlorophyll in the reaction 5 and reaction 13oils following silica treatment as described above.

TABLE 17 Composition of oils after enzyme treatment or after enzyme andsilica treatment Lovibond FFA Soap P Ca Mg Fe UV/Vis HPLC* Color (%)(ppm) (ppm) (ppm) (ppm) (ppm) (ppb) (ppm) Red Yellow ORSBO 0.12 20 0.30.2 b.d. b.d. 331 3.85 11.1 70 130 0.10 b.d. 0.2 0.1 b.d. b.d. 298 1.159.9 70 135 b.d. b.d. b.d. 0.1 b.d. b.d. 138 1.06 9.1 70 1310 n.m. b.d.b.d. 0.1 b.d. b.d. 90 1.05 9 70 1320 n.m. b.d. b.d. b.d. b.d. b.d. 411.03 8.6 70 132-2114 n.m. n.m. n.m. n.m. n.m. n.m. 104 1.01 8.3 70 b.d.= below detection n.m. = not measured *= HPLC was a measurement of totalchlorophyll derivatives

The results in Table 18 show the chlorophyll substrates and products inthe reaction 5 and 13 oils following silica treatment as describedabove.

TABLE 18 Chlorophyll substrate and product composition in oils afterenzyme treatment or after enzyme and silica treatment a a′ b CHYL PYNPPYN POB PPOB PYN POB CHYL PYN ppm ppm ppm ppm ppm ppm ppm ppm ppm ORSBO0.21 0.70 0.24 b.d. ND 1.26 b.d. 0.28 0.24 130 0.17 0.31 0.05 0.03 0.03b.d. b.d. 0.22 0.11 135 0.17 0.31 0.04 b.d. b.d. b.d. b.d. 0.21 0.111310 0.17 0.31 0.03 b.d. b.d. b.d. b.d. 0.22 0.11 1320 0.17 0.31 0.02b.d. b.d. b.d. b.d. 0.22 0.11 132-2114 0.16 0.27 0.03 b.d. b.d. 0.17b.d. 0.22 ND b b′ Decolorase PPYN POB PPOB CHYL PYN POB Total Sub. Prod.ppm ppm ppm ppm ppm ppm ppm ppm ppm ORSBO 0.49 b.d. b.d. b.d. 0.43 b.d.3.85 3.85 b.d. 130 0.23 b.d. b.d. b.d. b.d. b.d. 1.15 1.09 0.06 135 0.22b.d. b.d. b.d. b.d. b.d. 1.06 1.06 b.d. 1310 0.22 b.d. b.d. b.d. b.d.b.d. 1.05 1.05 b.d. 1320 0.22 b.d. b.d. b.d. b.d. b.d. 1.03 1.03 b.d.132-2114 0.16 b.d. b.d. b.d. b.d. b.d. 1.01 1.01 b.d. CHYL =Chlorophyll; PYN = Pheophytin; PPYN = Pyropheophytin; POB =Pheophorbide; PPOB = Pyropheophorbide; Sub = Substrates; Prod = Productsb.d. = below detection ND = not detected

In a direct comparison of SP-2114 and SP-2115, SP-2114 was not as goodas SP-2115, but was able to reduce the chlorophyll from 298 ppb in thereaction 13 oil to 104 ppb, as compared to 41 ppb achieved usingSP-2115, as reported from the UV/Vis method.

Reaction 14—Silica Treatment. Comparison of SP-2113, SP-2115, andSP-2119

Three 500 gram samples of the enzyme treated refined soybean oil fromreaction 14 were split and added to three 1000 mL round bottom flaskwith the configuration of Adsorbent procedure. The oils were heated to80° C. and 0.5, 1.0 and 2.0 grams of silica SP-2115 were mixed into theoil and a vacuum of approximately 100 mbar was added. The temperaturewas increased to 100° C. and mixed for 30 minutes. The vacuum was brokenand the material filter with a Buchner Funnel. The filter disc and cakewere a dark green color. The oils were labeled as 145, 1410, and 1420respectively. Oil labeled as 140 was the sample of reaction 14.

A 500 gram sample of the enzyme treated refined soybean oil fromreaction 14 was added to a 1000 mL round bottom flask with theconfiguration of Adsorbent procedure. The oil was heated to 80° C. and2.0 grams of silica SP-2113 was mixed into the oil and a vacuum ofapproximately 100 mbar was added. The temperature was increased to 100°C. and mixed for 30 minutes. No change in the color of the oil wasobserved during the adsorption process. The vacuum was broken and thematerial filter with a Buchner Funnel. The filter disc and cake wereyellow. The sample was labeled as 142-2113.

A 500 gram sample of the enzyme treated refined soybean oil fromreaction 14 was added to a 1000 mL round bottom flask with theconfiguration of Adsorbent procedure. The oil was heated to 80° C. and2.0 grams of silica SP-2119 was mixed into the oil and a vacuum ofapproximately 100 mbar was added. The temperature was increased to 100°C. and mixed for 30 minutes. No change in the color of the oil wasobserved during the adsorption process. The vacuum was broken and thematerial filter with a Buchner Funnel. The filter disc and cake wereyellow. The sample was labeled as 142-2119.

The once refined soybean oil enzyme treated and silica treated samples,1420 (471 grams) and 1410 (461 grams) were combined and placed in a 3 LClaisen flask and assembled according the deodorization procedure. Theoil was sparged with nitrogen for approximately 2 minutes. The vacuumwas initiated and the nitrogen sparge was discontinued and water vaporfrom the steam generator was allowed to begin the deodorization process.The vacuum achieved was between 0.28-0.56 mBar during the deodorizationprocess. The oil was heated under vacuum and water sparge (3 wt. %) to230° C. The sparge and temperature were maintained for two hours. Theheat was discontinued and the oil was allowed to cool under vacuum andwater sparge. The vacuum was broken with nitrogen at approximately 100°C. and allowed to further cool to 70° C. before opening to the air. Theoil was colorless with no green tint. The oil was labeled as14101420-DEO.

The results in Table 19 show the content of free fatty acids (FFA),soap, P, Ca, Mg, Fe, and chlorophyll in the reaction 14 oil followingsilica treatment as described above.

TABLE 19 Composition of ORSBOs after enzyme treatment or after enzymeand silica treatment Lovibond FFA Soap P Ca Mg Fe UV/Vis HPLC* Color (%)(ppm) (ppm) (ppm) (ppm) (ppm) (ppb) (ppm) Red Yellow ORSBO 0.06 27 1.00.4 tr b.d. 319 3.90 9.2 70 140 0.05 b.d. 0.2 0.1 b.d. b.d. 302 1.46 9.670 145 n.m. n.m. b.d. 0.1 b.d. b.d. n.m. 1.09 n.m. n.m. 1410 n.m. n.m.b.d. 0.1 b.d. b.d. 94 1.07 9.2 70 1420 n.m. n.m. b.d. 0.1 b.d. b.d. 521.04 8.7 70 142-2113 n.m. n.m. n.m. n.m. n.m. n.m. 309 1.13 8.7 70142-2119 n.m. n.m. n.m. n.m. n.m. n.m. 305 1.16 8.9 70 14101420-Deo 0.02n.m. n.m. n.m. n.m. n.m. 55 0.46 0.1 2.4 b.d. = below detection n.m. =not measured *= HPLC was a measurement of total chlorophyll derivatives

The results in Table 20 show the chlorophyll substrates and products inthe reaction 14 oil following silica treatment as described above.

TABLE 20 Chlorophyll substrate and product composition in ORSBOs afterenzyme treatment or after enzyme and silica treatment a a′ b CHYL PYNPPYN POB PPOB PYN POB CHYL PYN ppm ppm ppm ppm ppm ppm ppm ppm ppm ORSBO0.22 0.72 0.25 b.d. b.d. 1.27 b.d. 0.26 0.24 140 0.17 0.31 0.08 0.050.05 b.d. 0.03 0.22 0.11 145 0.17 0.31 0.05 b.d. b.d. b.d. b.d. 0.220.11 1410 0.17 0.31 0.03 b.d. b.d. b.d. b.d. 0.22 0.11 1420 0.17 0.310.02 b.d. b.d. b.d. b.d. 0.22 0.11 142-2113 0.16 0.25 0.08 b.d. b.d.0.18 b.d. 0.22 b.d. 142-2119 0.16 0.28 0.08 b.d. b.d. 0.18 b.d. 0.22b.d. 14101420-Deo 0.09 b.d. 0.02 b.d. b.d. b.d. b.d. 0.16 b.d. b b′Decolorase PPYN POB PPOB CHYL PYN POB Total Sub. Prod. ppm ppm ppm ppmppm ppm ppm ppm ppm ORSBO 0.52 b.d. b.d. b.d. 0.43 b.d. 3.90 3.90 b.d.140 0.23 0.03 0.05 0.11 b.d. b.d. 1.46 1.28 0.18 145 0.23 b.d. b.d. b.d.b.d. b.d. 1.09 1.09 b.d. 1410 0.23 b.d. b.d. b.d. b.d. b.d. 1.07 1.07b.d. 1420 0.22 b.d. b.d. b.d. b.d. b.d. 1.04 1.04 b.d. 142-2113 0.24b.d. b.d. b.d. b.d. b.d. 1.13 1.13 b.d. 142-2119 0.24 b.d. b.d. b.d.b.d. b.d. 1.16 1.16 b.d. 14101420-Deo 0.18 b.d. b.d. b.d. b.d. b.d. 0.460.46 b.d. CHYL = Chlorophyll; PYN = Pheophytin; PPYN = Pyropheophytin;POB = Pheophorbide; PPOB = Pyropheophorbide; Sub = Substrates; Prod =Products b.d. = below detection

Unlike samples treated with SP-2115, samples treated with SP-2113 orSP-2119 did not demonstrate any activity for the removal of chlorophyllas measured by the UV/Vis method. This method is accepted by theindustry.

Reaction 15—Silica Treatment and/or Deodorization

Three 500 g samples of the enzyme treated refined soybean oil fromreaction 15 were split and added to three 1000 mL round bottom flaskwith the configuration of Adsorbent procedure. The oils were heated to80° C. and 0.5, 1.0 and 2.0 grams of silica SP-2115 were mixed into theoil and a vacuum of approximately 100 mbar was added. The temperaturewas increased to 100° C. and mixed for 30 minutes. The vacuum was brokenand the material filter with a Buchner Funnel. The filter disc and cakewere a dark green color. The oils were labeled as 155, 1510, and 1520respectively. Oil labeled as 150 was the sample of reaction 15.

The once refined soybean oil enzyme treated and silica treated samples,1520 (457 grams) and 1510 (461 grams) were combined and placed in a 3 LClaisen flask and assembled according the deodorization procedure. Theoil was sparged with nitrogen for approximately 2 minutes. The vacuumwas initiated and the nitrogen sparge was discontinued and water vaporfrom the steam generator was allowed to begin the deodorization process.The vacuum achieved was between 0.57-0.97 mBar during the deodorizationprocess. The oil was heated under vacuum and water sparge (3 wt. %) to230° C. The sparge and temperature were maintained for two hours. Theheat was discontinued and the oil was allowed to cool under vacuum andwater sparge. The vacuum was broken with nitrogen at approximately 100°C. and allowed to further cool to 70° C. before opening to the air. Theoil was colorless with no green tint. The oil was labeled as15101520-DEO.

618 grams of the enzyme treated oil from reaction 15, without anyadsorbent treatment, was placed in a 3 L Claisen flask and assembledaccording the deodorization procedure. The oil was sparged with nitrogenfor approximately 2 minutes. The vacuum was initiated and the nitrogensparge was discontinued and water vapor from the steam generator wasallowed to begin the deodorization process. The vacuum achieved wasbetween 1.15-1.40 mBar during the deodorization process. The oil washeated under vacuum and water sparge (3 wt. %) to 230° C. The sparge andtemperature were maintained for two hours. The heat was discontinued andthe oil was allowed to cool under vacuum and water sparge. Broke vacuumwith nitrogen at 100° C. and allowed to cool to 70° C. before opening tothe air. The oil was colorless without any green tint. The oil waslabeled as 150-DEO.

The results in Table 21 show the contents of free fatty acids (FFA),soap, P, Ca, Mg, Fe, and chlorophyll in the reaction 15 oil followingdeodorization and/or silica treatment as described above.

TABLE 21 Composition of oils after enzyme treatment or after enzyme andsilica treatment Lovibond FFA Soap P Ca Mg Fe UV/Vis HPLC* Color (%)(ppm) (ppm) (ppm) (ppm) (ppm) (ppb) (ppm) Red Yellow Refined 0.03 2422.8 0.7 0.2 tr 321 3.90 n.m. n.m. 150 0.02 tr 0.3 0.2 b.d. 0.1 282 2.059.3 70 155 n.m. b.d. 0.1 0.2 b.d. b.d. 219 1.94 8.7 70 1510 n.m. b.d.0.1 0.1 b.d. b.d. 133 1.79 8.9 70 1520 n.m. b.d. 0.1 0.1 n.m. b.d. 331.04 8.4 70 15101520-Deo 0.02 n.m. n.m. n.m. n.m. n.m. 40 0.82 0.0 2.1150-Deo 0.02 n.m. n.m. n.m. n.m. n.m. 176 0.88 0.3 3.2 tr = trace b.d. =below detection n.m. = not measured *= HPLC was a measurement of totalchlorophyll derivatives

The results in Table 22 show the chlorophyll substrates and products inthe reaction 15 oil following deodorization and/or silica treatment asdescribed above. The results demonstrate the need for an adsorbent toremove the final lower color.

TABLE 22 Chlorophyll substrate and product composition in oils afterenzyme treatment or after enzyme and silica treatment a a′ b CHYL PYNPPYN POB PPOB PYN POB CHYL PYN ppm ppm ppm ppm ppm ppm ppm ppm ppmRefined 0.20 0.71 0.26 b.d. b.d. 1.27 b.d. 0.27 0.23 150 0.17 0.33 0.13b.d. b.d. 0.62 b.d. 0.22 0.11 155 0.18 0.32 0.10 b.d. b.d. 0.61 b.d.0.22 0.11 1510 0.17 0.32 0.05 b.d. b.d. 0.61 b.d. 0.22 0.11 1520 0.170.31 0.02 b.d. b.d. b.d. b.d. 0.22 0.11 15101520-Deo 0.10 0.31 0.03 b.d.b.d. b.d. b.d. 0.15 b.d. 150-Deo 0.06 0.31 0.13 b.d. b.d. b.d. b.d. 0.13b.d. b b′ Decolorase PPYN POB PPOB CHYL PYN POB Total Sub. Prod. ppm ppmppm ppm ppm ppm ppm ppm ppm Refined 0.52 b.d. b.d. b.d. 0.43 b.d. 3.903.90 b.d. 150 0.25 b.d. b.d. b.d. 0.21 b.d. 2.05 2.05 b.d. 155 0.25 b.d.b.d. b.d. b.d. b.d. 1.79 1.79 b.d. 1510 0.25 b.d. b.d. b.d. 0.21 b.d.1.94 1.94 b.d. 1520 0.22 b.d. b.d. b.d. b.d. b.d. 1.04 1.04 b.d.15101520-Deo 0.23 b.d. b.d. b.d. b.d. b.d. 0.82 0.82 b.d. 150-Deo 0.24b.d. b.d. b.d. b.d. b.d. 0.88 0.88 b.d. CHYL = Chlorophyll; PYN =Pheophytin; PPYN = Pyropheophytin; POB = Pheophorbide; PPOB =Pyropheophorbide; Sub = Substrates; Prod = Products b.d. = belowdetection

Reaction 16—Silica Treatment or Deodorization

Three 500 gram samples of the enzyme treated refined soybean oil fromreaction 16 were split and added to three 1000 mL round bottom flaskwith the configuration of Adsorbent procedure. The oils were heated to80° C. and 0.5, 1.0 and 2.0 grams of silica SP-2115 were mixed into theoil and a vacuum of approximately 100 mbar was added. The temperaturewas increased to 100° C. and mixed for 30 minutes. The vacuum was brokenand the material filter with a Buchner Funnel. The filter disc and cakewere a dark green color. The oils were labeled as 165, 1610, and 1620.The oil labeled as 160 was the sample from reaction 16.

825.3 grams of the enzyme treated oil, without any adsorbent treatment,was placed in a 3 L Claisen flask and assembled according thedeodorization procedure. The oil was sparged with nitrogen forapproximately 2 minutes. The vacuum was initiated and the nitrogensparge was discontinued and water vapor from the steam generator wasallowed to begin the deodorization process. The vacuum achieved wasbetween 1.15-1.40 mBar during the deodorization process. The oil washeated under vacuum and water sparge (3 wt. %) to 230° C. The sparge andtemperature were maintained for two hours. The heat was discontinued andthe oil was allowed to cool under vacuum and water sparge. Broke vacuumwith nitrogen at 100° C. and allowed to cool to 70° C. before opening tothe air. The color was expected to be greening, but was very slightlyred with no greenish tint. The sample was labeled as 160-Deo.

The results in Table 23 show the contents of free fatty acids (FFA),soap, P, Ca, Mg, Fe, and chlorophyll in the reaction 16 oil followingdeodorization or silica treatment as described above.

TABLE 23 Composition of oils after enzyme treatment or after enzyme andsilica treatment Lovibond FFA Soap P Ca Mg Fe UV/Vis HPLC* Color (%)(ppm) (ppm) (ppm) (ppm) (ppm) (ppb) (ppm) Red Yellow Refined 0.05 3963.3 0.9 0.2 b.d. 321 2.19 n.m. n.m. 160 0.03 tr b.d. 0.2 b.d. b.d. 3011.84 9.1 70 165 n.m. b.d. b.d. 0.2 b.d. b.d. 155 1.73 8.7 70 1610 n.m.n.m. 0.1 0.1 b.d. b.d. 79 n.m. 8.6 70 1620 n.m. n.m. b.d. 0.1 b.d. b.d.59 1.16 8.3 70 160-Deo 0.02 n.m. n.m. n.m. n.m. n.m. 210 0.94 0.2 3.3 tr= trace b.d. = below detection n.m. = not measured *= HPLC was ameasurement of total chlorophyll derivatives

The results in Table 24 show the chlorophyll substrates and products inthe reaction 16 oil following deodorization or silica treatment asdescribed above. The results demonstrate the need for treatment with anadsorbent for final green color removal.

TABLE 24 Chlorophyll substrate and product composition in oils afterenzyme treatment or after enzyme and silica treatment a a′ b CHYL PYNPPYN POB PPOB PYN POB CHYL PYN ppm ppm ppm ppm ppm ppm ppm ppm ppmRefined 0.21 0.36 0.12 b.d. b.d. 0.63 b.d. 0.28 0.12 160 0.17 0.32 0.100.04 0.03 0.61 b.d. 0.22 0.11 165 0.17 0.32 0.06 b.d. b.d. 0.61 b.d.0.22 0.11 1610 n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. 1620 0.170.31 0.02 b.d. b.d. ND b.d. 0.21 0.11 160-Deo 0.11 0.31 0.11 b.d. b.d.ND b.d. 0.17 ND b b′ Decolorase PPYN POB PPOB CHYL PYN POB Total Sub.Prod. ppm ppm ppm ppm ppm ppm ppm ppm ppm Refined 0.26 b.d. b.d. b.d.0.21 b.d. 2.19 2.19 b.d. 160 0.24 b.d. b.d. b.d. b.d. b.d. 1.84 1.770.07 165 0.24 b.d. b.d. b.d. b.d. b.d. 1.73 1.73 b.d. 1610 n.m. n.m.n.m. n.m. n.m. n.m. 1620 0.23 b.d. b.d. 0.11 b.d. b.d. 1.16 1.16 b.d.160-Deo 0.24 b.d. b.d. b.d. b.d. b.d. 0.94 0.94 b.d. CHYL = Chlorophyll;PYN = Pheophytin; PPYN = Pyropheophytin; POB = Pheophorbide; PPOB =Pyropheophorbide; Sub = Substrates; Prod = Products b.d. = belowdetection ND = not detected

Example 10 Incubation of Once Refined Canola (ORCAN) Oil with the CHL26Enzyme, and Treatment with Silica SP-2115 or Commercial Silica orBleaching Clay

A five gallon plastic pail of Once Refined Canola (ORCAN) oil was mixedwith a high shear mixer to make uniform. Samples were pulled to use inthe following experiments.

Reaction 17—CHL26

3,000 grams of the once refined canola oil was placed into a 4 literglass beaker on a hot plate with overhead mixing. The oil was heated to60° C. under agitation. Once the material reached 60° C., the oil wasmoved to the shear mixer. 15 g of the decolorase enzyme CHL26 (preparedas described in Example 4) and 150 g of deionized water were added. Thematerial was shear mixed for 1 minute. The glass beaker was moved backto the overhead mixer and covered with plastic wrap. The oil was mixedfor 4 hours at 60° C. The oil temperature was increased to 75° C., andthe oil was centrifuged utilizing a Gyro-Centrifuge with a bowl withholes closed. Oil and heavy samples were collected.

The oil and heavy phase remaining in the bowl were poured into a 400 mLbeaker where the oil was decanted off. The remaining oil and heavy phasewere placed into 50 mL centrifuge tubes and spun. The oil remaining inthe tubes was discarded and the liquid heavy phases were combined. Theheavy phase was a dark green.

Reactions 18-20—CHL26

3000 grams of the once refined canola oil was placed into a 4 literjacket glass beaker. The temperature was set at 60° C. Once the materialreached 60° C., the oil was moved to a shear mixer. 15 g of thedecolorase enzyme CHL26 (prepared as described in Example 4) and 150 gof deionized water were added to the hot oil. The material was shearmixed for 1 minute. The glass beaker was moved back to the overheadmixer and covered again with plastic wrap. The oil was mixed for 4 hoursat 60° C. The oil temperature was increased to 75° C., and the oil wascentrifuged utilizing a Gyro-Centrifuge with a bowl with holes closed.The procedure was repeated three times and the oil was collected andcombined for reactions 18-20.

Reaction 17—Silica Treatment with SP-2115

500 grams of once refined canola oil from Reaction 17 was added to a1000 mL round bottom flask with the equipment configuration of above.The oil was heated to 80° C. and 2.0 g of silica SP-2115 was mixed intothe oil and a vacuum of approximately 100 mbar was added. Thetemperature was increased to 100° C. and mixed for 30 minutes. Thevacuum was broken and the material filter with a Buchner Funnel. Thisexperiment was repeated under the same conditions, except using 4, 6,and 8 grams of silica SP-2115.

Reactions 18-20—Silica Treatment. Comparison of SP-2115 and CommercialSilica and Bleaching Clay

The once refined canola oil from the combined reactions 18-20 wastreated with a commercially available silica (TriSyl® 300), a bleachingclay (Clariant 126FF), or two separate lots of SP-2115, as describedbelow.

500 grams of once refined canola oil from the combined reactions 18-20was added to a 1000 mL round bottom flask with the equipmentconfiguration of above. The oil was heated to 80° C. and 2.0 g ofTriSyl® 300 was mixed into the oil and a vacuum of approximately 100mbar was added. The temperature was increased to 100° C. and the oil wasmixed for 30 minutes. The vacuum was broken and the material filter witha Buchner Funnel.

500 grams of once refined canola oil from the combined reactions 18-20was added to a 1000 mL round bottom flask with the equipmentconfiguration of above. The oil was heated to 80° C. and 2.0 g of thebleaching clay Tonsil® supreme 126 FF(Clariant) was mixed into the oiland a vacuum of approximately 100 mbar was added. The temperature wasincreased to 100° C. and the oil was mixed for 30 minutes. The vacuumwas broken and the material filter with a Buchner Funnel.

500 grams of once refined canola oil from combined reactions 18-20 wasadded to a 1000 mL round bottom flask with the equipment configurationof above. The oil was heated to 80° C. and 2.0 g of the first lot ofsilica SP-2115 was mixed into the oil and a vacuum of approximately 100mbar was added. The temperature was increased to 100° C. and the oil wasmixed for 30 minutes. The vacuum was broken and the material filter witha Buchner Funnel.

500 grams of once refined canola oil from combined reactions 18-20 wasadded to a 1000 mL round bottom flask with the equipment configurationof above. The oil was heated to 80° C. and 2.0 g of the second lot ofsilica SP-2115 was mixed into the oil and a vacuum of approximately 100mbar was added. The temperature was increased to 100° C. and the oil wasmixed for 30 minutes. The vacuum was broken and the material filter witha Buchner Funnel.

The results in Table 25 show the contents of free fatty acids (FFA),soap, P, Ca, Mg, Fe, and sodium (Na) in the reaction 17 oil or thecombined reaction 18-20 oil following treatment with silica SP-2115,TriSyl® silica, or bleaching clay, as described above.

TABLE 25 Composition of oils after treatment with CHL26 or aftertreatment with CHL26 and SP-2115, TriSyl ® 300 silica, or bleaching claySoap FFA P Ca Mg Fe Na Oil (ppm) (%) (ppm) (ppm) (ppm) (ppm) (ppm) OnceRefined Can 171 0.09 4.8 1.1 0.2 0.10 11.4 Rxn 17 - CHL26 Can b.d. 0.051.0 1.9 tr 0.05 b.d. Rxn 17 - 2.0 g SP- Can n.m. 0.05 b.d. 0.5 b.d. 0.02b.d. 2115 Rxn 17 - 4.0 g SP- Can n.m. 0.05 tr 0.2 b.d. 0.03 b.d. 2115Rxn 17 - 6.0 g SP- Can n.m. 0.04 0.1 0.2 b.d. 0.02 b.d. 2115 Rxn 17 --8.0 g SP- Can n.m. 0.04 0.3 0.3 b.d. tr b.d. 2115 Rxn 18-20 -- CHL26 Canb.d. 0.05 0.8 2.0 tr 0.12 b.d  Rxn 18-20 - 2.0 g Can n.m. 0.05 b.d. 0.4b.d. b.d. b.d. TriSyl ®300 Rxn 18-20 - 2.0 g Can n.m. 0.06 0.2 0.9 b.d.0.04 b.d. Clariant 126FF Rxn 18-20 - 2.0 g Can n.m. 0.05 0.8 0.9 b.d.b.d. b.d. SP-2115 (2^(nd) lot) Rxn 18-20 - 2.0 g Can n.m. 0.05 0.7 0.6b.d. 0.05 b.d. SP-2115 (1^(st) lot) b.d.—below detection tr—tracen.m.—not measured Once Refined means washed and dried Can—canola

The oil samples (˜1 gram) were diluted in 100 ml volumetric flask withCHCl₃ (chloroform) and measured for chlorophyll content using the UV-Vismethod looking at the peak absorbance at 670 nm. Measurements were alsomade using the HPLC method. The results are set forth in Table 26.

TABLE 26 Chlorophyll content of oils after treatment with CHL26 or aftertreatment with CHL26 and SP-2115, TriSyl ® silica, or bleaching clayUV/Vis HPLC* Oil (ppb) (ppb) Starting Material (ORCO) 32733 36856 Rxn 1730781 27090 Rxn 17, 2 g SP-2115 16527 16722 Rxn 17, 4 g SP-2115 1449013680 Rxn 17, 6 g SP-2115 4524 5652 Rxn 17, 8 g SP-2115 3165 4812 Rxn18-20 Combined 31070 28840 Rxn 18-20, 2 g TriSyl 300 27881 25888 Rxn18-20, 2 g Clariant 126 FF 11975 6551 Rxn 18-20, 2 g SP-2115 (2^(nd)lot) 15462 13207 Rxn 18-20, 2 g SP-2115 (1^(st) lot) 18487 15595 *= HPLCwas a measurement of total chlorophyll derivatives

The results in Tables 27-28 show the chlorophyll substrates and productsin the reaction 17 or combined reaction 18-20 oils after treatment asdescribed above. The remaining levels are close to the level ofchlorophyll substrates and products needed in an industrial process.Optimizing the reaction conditions for the decolorase enzyme will enablethe elimination of bleaching earth for very green canola oils.

TABLE 27 Chlorophyll substrate and product composition in oils aftertreatment with CHL26 or after treatment with CHL26 and SP-2115, TriSyl ®300, or bleaching clay a a′ b b′ CHYL PYN PPYN POB PPOB PYN POB CHYL PYNPPYN POB PPOB CHYL PYN POB Total ppm ppm ppm ppm ppm ppm ppm ppm ppm ppmppm ppm ppm ppm ppm ppm Starting 0.41 11.83 13.76 b.d. b.d. 2.87 b.d.0.21 3.21 3.71 b.d. b.d. b.d. 0.86 b.d. 36.86 Material (ORCO) Rxn 17b.d. 2.95 6.85 4.52 2.76 2.90 0.58 0.19 0.88 3.77 0.97 0.04 b.d. 0.600.09 27.09 Rxn 17-2 g 0.12 3.11 5.88 0.19 b.d. 2.16 0.12 0.21 0.81 3.52b.d. b.d. b.d. 0.60 b.d. 16.72 SP-2115 Rxn 17-4 g 0.11 2.48 5.28 0.07b.d. 1.78 0.06 0.17 0.60 2.69 b.d. b.d. b.d. 0.44 b.d. 13.68 SP-2115 Rxn17-6 g 0.13 1.00 0.68 b.d. b.d. 0.94 b.d. 0.18 0.43 1.95 b.d. b.d. b.d.0.35 b.d. 5.65 SP-2115 Rxn 17-8 g 0.10 0.80 0.48 b.d. b.d. 0.94 b.d.0.19 0.37 1.62 b.d. b.d. b.d. 0.32 b.d. 4.81 SP-2115 Rxn 18-20 b.d. 2.806.94 4.74 2.74 2.44 0.61 0.18 0.83 3.70 1.51 1.61 b.d. 0.52 0.19 28.84combined Rxn 18-20 - b.d. 2.33 7.09 3.53 1.90 3.42 0.45 0.18 0.67 3.660.96 0.88 b.d. 0.69 0.12 25.89 TriSyl ® 300 Rxn 18-20 - b.d. 1.58 1.830.57 0.11 b.d. 0.12 0.18 0.63 0.18 0.40 0.37 b.d. 0.47 0.10 6.55Clariant 126 FF Rxn 18-20 - 0.10 2.74 5.27 0.15 0.11 b.d. b.d. 0.19 0.743.43 b.d. b.d. b.d. 0.49 b.d. 13.21 2 g SP-2115 (2nd lot) Rxn 18-20 -0.10 3.06 7.08 0.10 0.07 b.d. b.d. 0.19 0.74 3.78 b.d. b.d. b.d. 0.47b.d. 15.60 2 g SP-2115 (1st lot) CHYL = Chlorophyll; PYN = Pheophytin;PPYN = Pyropheophytin; POB = Pheophorbide; PPOB = Pyropheophorbide; Sub= Substrates; Prod = Products b.d. = below detection

TABLE 28 Chlorophyll substrate and product composition in oils aftertreatment with CHL26 or after treatment with CHL26 and SP-2115, TriSyl ®300, or bleaching clay. Decolorase Substrates Products (ppm) (ppm)Starting Material (ORCO) 36.86 b.d. Rxn 17 18.14 8.95 Rxn 17, 2 gSP-2115 16.41 0.31 Rxn 17, 4 g SP-2115 13.55 0.13 Rxn 17, 6 g SP-21155.65 b.d. Rxn 17, 8 g SP-2115 4.81 b.d. Rxn 18-20 combined 17.41 11.43Rxn 18-20, TriSyl ® 300 18.03 7.85 Rxn 18-20, Clariant 126 FF 4.87 1.68Rxn 18-20, 2 g SP-2115 (2^(nd) 12.96 0.25 lot) Rxn 18-20, 2 g SP-2115(1^(st) 15.42 0.17 lot)

The commercial silica “Trisyl® 300 has a limited ability to remove theproducts generated in the decolorase reactions, and actually appears toconvert some of the chlorophyll products back into substrates. Thebleaching earth has a greater ability to remove the chlorophyllsubstrates found in unreacted decolorase oils, but does not remove theproducts of the decolorase treated oils as well as the silicas of thepresent disclosure.

Example 11 Preparation of Silica Adsorbents

This example describes the preparation of the adsorbents in Reactions21-30 below:

Reaction 21—Preparation of SP-2113

600 grams of a TRISYL® silica, was dried at 60° C. to remove 173 gramsof water. The silica was then impregnated with a sodium hydroxidesolution containing 18.6 grams of NaOH and 81.9 grams of water. Thematerial was blended in a Waring blender for 5 minutes.

Reaction 22—Preparation of SP-2114

600 grams of TRISYL® silica, was blended in a Waring blender for 5minutes with 12.2 g of MgO powder.

Reaction 23—Preparation of SP-2115

600 grams of TRISYL® silica was blended in a Waring blender for 5minutes with 31.6 g of MgO powder.

Reaction 24—Preparation of SP-2116

600 grams of TRISYL® 300 silica, was dried at 60° C. to remove 173 gramsof water. The silica was then impregnated with a sodium hydroxidesolution containing 11.4 grams of NaOH and 92 grams of water. Thematerial was blended in a Waring blender for 5 minutes.

Reaction 25—Preparation of SP-2117

600 grams of TRISYL® 300 silica was dried at 60° C. to remove 173 gramsof water. The silica was then impregnated with a sodium hydroxidesolution containing 17.8 grams of NaOH and 92 grams of water. Thematerial was blended in a Waring blender for 5 minutes.

Reaction 26—Preparation of SP-2119

600 grams of a silica xerogel containing less than 10 wt % water, asurface area of 707 m²/g and a median particle size of 19 microns, wasblended in a Waring blender with 30 grams of MgO for 5 minutes.

Reaction 27—Preparation of Adsorbent A

4.8 grams of SP-2115 was dried in an oven at 110° C. for 3 hours.

Reaction 28—Preparation of Adsorbent B

5 grams of MgO powder and 20 grams TRISYL® silica were blended into acontainer, sealed, then mixed by shaking for 1 hour.

Reaction 29—Preparation of Adsorbent C

24.5 grams of a silica xerogel containing less than 10 wt % water, andhaving a surface area of 707 m²/g and a median particle size of 19microns, was impregnated with 23.6 grams of DI water then added to acontainer containing 2.5 grams of MgO powder. The contents were sealedand mixed by shaking for 1 hour.

Reaction 30—Preparation of Adsorbent D

264 grams of silica xerogel with 4 wt % water, a surface area of 330m²/g, and a particle size between 88 and 210 microns was impregnatedwith 303 grams of DI water. The material was divided into six differentcontainers, each containing 6 grams of MgO powder. Each container wasmixed by shaking for 1 hour, then the contents were all combined into alarger container and blended by shaking for 1 hour.

Example 12 Enzymatic and Adsorbent Treatment of Oils

Adsorbents prepared in Example 11 were used to further treat two batchesof oil previously subjected to decolorase treatment (i.e., oil 11120,prepared as described in Example 9).

Reaction 31—Treatment of Decolorase Treated Oil with Adsorbent A

100 grams of enzymatically treated oil 11120 was heated to 80° C. then0.21 grams of Adsorbent A was mixed into the oil. A 100 mbar vacuum wasapplied then the temperature was set to 100° C. and mixed for 30minutes. The vacuum was broken and the material was filtered with aBuchner Funnel. The filter disc and cake were dark green.

Reaction 32—Treatment of Decolorase Treated Oil with Adsorbent B

100 grams of enzymatically treated oil 11120 was heated to 80° C. then0.4 grams of Adsorbent B was mixed into the oil. A 100 mbar vacuum wasapplied then the temperature was set to 100° C. and mixed for 30minutes. The vacuum was broken and the material was filtered with aBuchner Funnel. The filter disc and cake were dark green.

Reaction 33—Treatment of Decolorase Treated Oil with Adsorbent C

100 grams of enzymatically treated oil 11120 was heated to 80° C. then0.4 grams of Adsorbent C was mixed into the oil. A 100 mbar vacuum wasapplied then the temperature was set to 100° C. and mixed for 30minutes. The vacuum was broken and the material was filtered with aBuchner Funnel. The filter disc and cake were dark green.

Reaction 34—Treatment of Decolorase Treated Oil with Adsorbent D

100 grams of enzymatically treated oil 11120 was heated to 80° C. then0.4 grams of Adsorbent D was mixed into the oil. A 100 mbar vacuum wasapplied then the temperature was set to 100° C. and mixed for 30minutes. The vacuum was broken and the material was filtered with aBuchner Funnel. The filter disc and cake were dark green.

Reaction 35—Treatment of Decolorase Treated Oil with TRISYL® Silica

100 grams of enzymatically treated oil 11120 was heated to 80° C. then0.4 grams of TRISYL® silica was mixed into the oil. A 100 mbar vacuumwas applied then the temperature was set to 100° C. and mixed for 30minutes. The vacuum was broken and the material was filtered with aBuchner Funnel. The filter disc and cake were green.

Reaction 36—Treatment of Decolorase Treated Oil with TRISYL® 300 Silica

100 grams of enzymatically treated oil 11120 was heated to 80° C. then0.4 grams of TRISYL® 300 silica was mixed into the oil. A 100 mbarvacuum was applied then the temperature was set to 100° C. and mixed for30 minutes. The vacuum was broken and the material was filtered with aBuchner Funnel. The filter disc and cake were yellow.

The green color concentrations from the oils produced in Reactions 31-36were determined using the AOCS UV/Vis method. The results are set forthin Table 29.

TABLE 29 Green color of decolorase treated oil after further treatmentwith various adsorbents. Reaction Green color (ppm) First batch of oil11120 11.4 31 0.2% of Adsorbent A 6.5 32 0.4% of Adsorbent B 5.7 33 0.4%of Adsorbent C 5.6 34 0.4% of Adsorbent D 7.0 Second batch of oil 1112018.9 35 0.4% of TRISYL ® silica 18.6 36 0.4% TRISYL ® 300 silica 18.8

1. An adsorbent for removing chlorophyll and chlorophyll derivativesfrom an oil, which adsorbent comprises an amorphous porous silica geltreated with an alkaline earth metal oxide, said adsorbent having a pHof from about 7 or greater, an alkaline earth metal oxide content of atleast 0.1 weight percent (wt %), on a dry basis, and a water content offrom about 3 wt % or greater.
 2. The adsorbent of claim 1, wherein theadsorbent comprises from 1 to about 40 wt % alkaline earth metal oxide,on a dry basis.
 3. The adsorbent of claim wherein the alkaline earthmetal oxide is selected from the group consisting of magnesium oxide,calcium oxide, barium oxide, beryllium oxide, or combinations thereof.4. The adsorbent of claim wherein the alkaline earth metal oxide ismagnesium oxide.
 5. The adsorbent of claim wherein the amount ofmagnesium oxide ranges from about 5 wt % to about 25 wt % MgO, whereinthe magnesium oxide preferably comprises from about 10 to about 20 wt %MgO.
 6. (canceled)
 7. The adsorbent of claim 1, wherein the adsorbenthas a pH of from 7.0 to 10.0, preferably from 7.5 to 9.7, more preferredfrom 8.0 to 9.5. 8-9. (canceled)
 10. The adsorbent of claim 1, whereinthe adsorbent has a water content of 10 wt % or greater, preferably fromabout 25 to about 75 wt %, more preferred from about 40 to about 70 wt%, still more preferred from about 55 to about 65 wt %. 11-13.(canceled)
 14. The adsorbent of claim 1, wherein the adsorbent has amedian particle size of from about 0.1 to about 2,000 microns,preferably from about 2 to about 500 microns, more preferred from about5 to about 50 microns. 15-16. (canceled)
 17. The adsorbent of claim 1,wherein the adsorbent has a surface area of at least 50 m²/g, preferablyfrom about 50 to about 800 m²/g.
 18. (canceled)
 19. The adsorbent ofclaim 1, wherein the adsorbent has a pore volume of at least about 0.1cc/g, preferably of at least about 0.4 cc/g, more preferred from about0.7 to about 2.0 cc/g. 20-21. (canceled)
 22. The adsorbent of claimwherein the adsorbent has a pH of from about 7 to about 10, a magnesiumoxide amount of from about 5 to 25 wt % MgO, on a dry basis, and a watercontent of from about 25 wt % to about 65 wt %, preferably has a pH offrom about 8 to about 9.5, a magnesium oxide amount of from about 10 to20 wt % MgO, on a dry basis, and a water content of from about 55 wt %to about 65 wt %.
 23. (canceled)
 24. The adsorbent of claim 1, whereinthe adsorbent has the capability of removing chlorophyll and chlorophyllderivatives from an oil, wherein the oil preferably comprises atriacylglycerol-based oil.
 25. (canceled)
 26. The adsorbent of claim 24,wherein the triacylglycerol-based oil is selected from the groupconsisting of canola oil, castor oil, coconut oil, coriander oil, cornoil, cottonseed oil, hazelnut oil, hempseed oil, linseed oil, mangokernel oil, meadowfoam oil, neat's foot oil, olive oil, palm oil, palmkernel oil, palm olein, peanut oil, rapeseed oil, rice bran oil,safflower oil, sasanqua oil, sesame oil, soybean oil, sunflower seedoil, tall oil, tsubaki oil, vegetable oil, and an oil from alga.
 27. Theadsorbent of claim 24, wherein the oil is a decolorase-treated oil. 28.The adsorbent of claim 27, wherein the silica gel is a hydrogel or ahydrated xerogel.
 29. The adsorbent of claim 28, wherein the adsorbentreduces the total concentration of chlorophyll and chlorophyllderivatives in the decolorase-treated oil by at least 5% by weight,compared to the total concentration of chlorophyll and chlorophyllderivatives in the decolorase-treated oil prior to contact with theadsorbent, preferably by at least 50% by weight, compared to the totalconcentration of chlorophyll and chlorophyll derivatives in thedecolorase-treated oil prior to contact with the adsorbent. 30.(canceled)